Molecule assigning genotype to phenotype and use thereof

ABSTRACT

A molecule assigning a genotype to a phenotype, which comprises a nucleic acid portion having a nucleotide sequence reflecting the genotype, and a protein portion comprising a protein involved in exhibition of the phenotype, a 3′-terminal end of the nucleic acid portion and a C-terminal end of the protein portion being covalently bound, and a method for constructing the molecule for assigning the genotype to the phenotype, which comprises (a) preparing a DNA containing a gene which has no termination codon, (b) transcribing the prepared DNA into RNA, (c) bonding a chimeric spacer composed of DNA and RNA to a 3′-terminal end of the obtained RNA, (d) bonding, to a 3′-terminal end of the obtained bonded product, a nucleoside or a substance having a chemical structure analogous to that of a nucleoside, which can be covalently bound to an amino acid or a substance having a chemical structure analogous to that of an amino acid, and (e) performing protein synthesis in a cell-free protein synthesis system using the obtained bonded product as mRNA to bond a nucleic acid portion containing the gene to a translation product of the gene. The molecule assigning the genotype to the phenotype of the present invention is an extremely useful substance that can be used for evolutionary molecular engineering, i.e., modification of functional biopolymers such as enzymes, antibodies, and ribozymes, and creation of biopolymers having functions which cannot be found in living organisms.

TECHNICAL FIELD

[0001] The present invention relates to a molecule assigning a genotypeto a phenotype. More specifically, it relates to a molecule assigning agenotype to a phenotype, comprising a nucleic acid portion having anucleotide sequence reflecting the genotype and a protein portioncomprising a protein involved in exhibition of the phenotype. Themolecule assigning the genotype to the phenotype of the presentinvention is a highly useful substance that can be utilized inevolutionary molecular engineering such as in the modification ofenzymes, antibodies, ribozymes and other such functional biopolymers andcreation of biopolymers having functions not found in living organisms.

[0002] Through advances in biochemistry, molecular biology andbiophysics, it has been learned that living organisms are molecularmachines which function and propagate by interactions among molecules.Among the characteristics of earth's living organisms, the fundamentalsare their preservation of genetic information in DNA nucleotidesequences and their ability to translate this information intofunctional proteins through the medium of mRNA. Owing to progress ingenetic engineering, biopolymers with given sequences, like nucleotidesand peptides, can now be easily synthesized. Protein engineering and RNAengineering, today a focus of attention, owe their existence to geneticengineering. The aims of protein engineering and RNA engineering are tosolve the puzzle of the three-dimensional structures required forproteins and RNA fulfilling specific functions and to enable humans tofreely design proteins and RNA possessing desired functions. Because ofthe diversity and complexity of these structures and the difficulty of atheoretical approach to their three-dimensional structures, however,current protein engineering and RNA engineering are still at the stageof modifying some of residues at active sites and observing changes inthe structure and functions. Human knowledge has thus not yet reachedthe stage of designing proteins and RNA.

[0003] Understanding the functions of biopolymers in their relationshipto the elemental processes of higher life phenomena will requireelucidation of the correlation between protein molecular structure andfunction. The line of thought we take in the following is not only tomake the best of “human knowledge” but also to take advantage of the“wisdom of nature.” This is because we concluded that we would have toacquire the ability to put both to work in order to overcome the currentdifficulties of protein engineering and move forward with the design andproduction of functional biopolymers. When the classical methods arediverted to the design of proteins with new functions and activities,the difficulty of protein design by site-specific mutations cansometimes be avoided. This can be called “taking advantage of the wisdomof nature.”

[0004] Although the drawback of this method is the difficulty ofscreening to identify mutants with new functions and activities, thisdifficulty is overcome by the RNA catalysts that have recently come intothe spotlight. Attempts have been made to select an RNA with specificcharacteristics from among RNAs synthesized to have an extremely largenumber of random sequences (about 10¹³ types) (Ellington, A. D. &Szostak, J. W. (1990) Nature, 346, 818-822).

[0005] This is an example of evolutionary molecular engineering. Astypified by this example, the primary goal in the evolutionary molecularengineering of proteins is to find out optimum sequences by searching anexpansive sequence space of a scale unimaginable in conventional proteinengineering. By “making the best of human knowledge” to devise ascreening system for this, it will be possible to discover numerousquasi-optimum sequences around the optimum sequences and thus toconstruct an experimental system for studying “sequence vs function.”

[0006] The remarkable functions of living bodies were acquired throughthe process of evolution. Therefore, if evolution can be replicated, itshould be possible to modify enzymes, antibodies, ribozymes and otherfunctional biopolymers and, further, to create biopolymers withfunctions not found in living organisms in the laboratory. Needless tosay, research on protein modification and creation is an object ofutmost importance to various aspects of biotechnology such asutilization of enzymes as industrial catalysts, biochips, biosensors andsugar-chain engineering.

[0007] Given the fact that molecular design utilizing structural theoryis, as symbolized by the continuing high regard for “screening,” stillin an unperfected state, the evolutionary technique has a practicalvalue for utilization in selecting useful proteins as a more efficientstrategy. Building a “time machine” capable of more efficientlyproducing evolution in a laboratory, if such were possible, would notonly enable modification of enzymes, antibodies (vaccines, monoclonalantibodies etc.) and other existing proteins but also open the way tothe creation of enzymes for decomposing environmental contaminants,purifiers and others and new proteins not present in the biologicalworld. If an experimental system for protein evolution can beestablished, therefore, it can be expected to be aggressively utilizablefor application in a wide range of fields including power saving andenergy preservation in industrial processes, energy production andenvironmental preservation. The assigning molecule of the presentinvention is a highly useful substance in protein modification and otheraspects of evolutionary molecular engineering.

BACKGROUND ART

[0008] Evolutionary molecular engineering is a field of study thatattempts to conduct molecular design of functional polymers by utilizinghigh-speed molecular evolution in the laboratory, i.e., by laboratoryinvestigation and optimization of the adaptive locomotion of biopolymersin sequence space. It is a completely new molecular biotechnology thatfirst produced substantial results in 1990 (Yuzuru Husimi (1991) Kagaku,61, 333-340; Yuzuru Husimi (1992) Koza Shinka, Vol. 6, University ofTokyo Publishing Society).

[0009] Life is a product of molecular evolution and natural selection.The evolution of molecules is a universal life phenomenon but itsmechanism is not something that can be elucidated by studies that trackthe history of past evolution. Rather, the approach of constructing andstudying the behavior of simple molecules and life systems that evolvein the laboratory better provides fundamental knowledge regardingmolecular evolution and enables establishment of a verifiable theoryapplicable in molecular engineering.

[0010] It is known that a polymer system will evolve if it satisfies thefollowing five conditions: (1) an open system far out of equilibrium,(2) a self-replicative system, (3) a mutation system, (4) a system withgenotype and phenotype assignment strategy, and (5) a system withappropriate adaptation topography in sequence space. (1) and (2) areconditions for occurrence of natural selection and (5) is determinedbeforehand by the physicochemical properties of the biopolymer. Thegenotype and phenotype assignment of (4) is a prerequisite for evolutionby natural selection.

[0011] The following three strategies are adopted in both the naturalworld and evolutionary molecular engineering: (a) ribozyme-type in whichthe genotype and the phenotype are carried on the same molecule, (b)virus-type in which the genotype and the phenotype form a complex, and(c) a cell-type in which the genotype and the phenotype are contained ina single compartment (FIG. 1).

[0012] As the ribozyme-type (a) in which the genotype and the phenotypeare carried on the same molecule is a simple system, success with RNAcatalysts (ribozymes) has already been reported (Hiroshi Yanagawa (1993)New Age of RNA, pp.55-77, Yodosha).

[0013] Conceivable problem points of the cell-type (c) are (1) theaveraging effect, (2) the eccentricity effect and (3) the randomreplication effect. The averaging effect arises because the assignmentof the genotype to the phenotype statistically averages out and becomesambiguous when the number of copies of the cell genome is large. Sincean evolved genome is only one among the number of copies in a cell (n),performance enhancement averages out and a struggle for existence in thecell population begins at selection coefficient (s)/n. A smaller copynumber (n) is therefore advantageous for the cell-type. Due to thepresence of the eccentricity effect, however, when the number ofsegments is large, n must be very large to prevent the eccentricityeffect. The apparent selection coefficient in the struggle for existencein the cell population can therefore be expected to be very much smallerthan in the case of the virus-type. Since the time required forselection is proportional to the reciprocal of the selectioncoefficient, the rate of evolution is much slower than that of thevirus-type. Further, the random replication effect (3) is fatal to thecell-type. This is because the random replication of segmented essentialgenes by this effect makes replication of all essential genes prior tocell division extremely difficult. This means that even if an essentialgene with an advantageous mutation should occur, the probability of itsbeing replicated and passed on to a daughter cell is extremely low.

[0014] Uniting of the genotype and the phenotype as in the virus-type(b) is necessary for efficient evolution. various techniques havealready been proposed and are in the process of development forevolutionary molecular engineering of the virus-type (b) forming acomplex of the genotype and the phenotype, including phage display(Smith, G. P. (1985) Science 228, 1315-1317; Scott, J. K. & Smith, G. P.(1990) Science 249, 386-390), polysome display (Mattheakis, L. C. et al.(1994) Proc. Natl. Acad. Sci. USA 91, 9022-9026), encoded combinatoriallibrary (Brenner, S. & Lerner, R. A. (1992) Proc. Natl. Acad. Sci. USA89, 5381-5383), and cellstat (Husimi, Y. et al. (1982) Rev. Sci.Instrum. 53, 517-522).

[0015] Despite the importance of the magnitude of the searchablesequence space in evolutionary molecular engineering, however, a methodfor globally searching a sequence space comparable to that of theribozyme type has not yet been established for the virus-type.

[0016] The reason for this is that viruses currently used in the methodsuch as phage displays are parasites of existing cells and are thereforeunavoidably subject to restraints imposed by the host cells, among whichcan be listed: (1) that only a limited sequence space can be searchedowing to restriction by the cells, (2) membrane permeability, (3) biasdue to host, and (4) limitation on library owing to host population.

[0017] The polysome display method (Mattheakis, L. C. & Dower, W. J.(1995) WO95/11922) joins a nucleic acid and a protein via a ribosome bynon-covalent bonding. It is therefore suitable when the chain length atthe peptide position is short but encounters handling problems when thechain length is long as a protein. Since the huge ribosome remainsinterposed, the conditions at the time of the selection operation (e.g.,adsorption, elution or the like) are subjected to severe restriction.The encoded combinatorial library (Janda, F. H. & Lerner, R. A. (1996)WO96/22391) assigns a chemically synthesized peptide to a nucleic acidtag via beads. Since the yield of chemical synthesis of proteins witharound 100 residues is extremely poor using currently availabletechnologies, however, this technique can be used with shortchain-length peptides but not with long chain-length proteins.

[0018] One conceivable method of overcoming these problems is use of acell-free translation system. A virus-type strategy molecule that simplybinds the genotype and the phenotype in the cell-free systems has anumber of advantages including the following: (1) that a huge mutantpopulation approaching that of the ribozyme-type can be synthesized, (2)creation of various proteins without dependence on a host, (3) noproblem regarding membrane permeability, and (4) that the 21st code canbe used to introduce a non-native amino acid.

DESCRIPTION OF THE INVENTION

[0019] An object of the present invention is to provide a moleculecomprising a virus-type operation replicon which has the advantages ofthe aforementioned virus-type strategy molecule, exhibits a higherefficiency than phages, and suffers fewer limitations concerningenvironmental condition setting, namely, a molecule which should becalled “in vitro virus”, wherein a nucleic acid and a protein are boundby a chemical bond, that is, a molecule in which a genotype is assignedto a phenotype. More specifically, the present invention has beenaccomplished in order to provide a molecule exhibiting one-on-onerelationship between information and function, which can be utilized forcreation of functional proteins and peptides, by performing genotype(nucleic acid) assignment to phenotype (protein) using a cell-freeprotein synthesis system, and binding the 3′-terminal end of a gene tothe C-terminal end of a protein with a covalent bond on ribosome.Further, it is also an object of the present invention to obtain targetfunctional proteins or peptides through investigation of vast sequencespace, which is performed by repetition of selection of molecules thatassign genotypes to phenotypes formed as described above (also referredto as “in vitro virus” hereinafter) by the in vitro selection method,and amplification of gene portions of the selected in vitro viruses bythe reverse transcription PCR, and further amplification whileintroducing mutations.

[0020] The present inventors earnestly conducted investigations toachieve the aforementioned objects, and as a result, they found that twokinds of molecules that assign a genotype to a phenotype, comprising anucleic acid and a protein which were chemically bound can beconstructed on a ribosome in a cell-free protein synthesis system. Theyfurther found that a protein evolution simulation system can beconstructed wherein the assigning molecules (in vitro viruses) wereselected by the in vitro selection method, gene portions of the selectedin vitro viruses were amplified by reverse transcription PCR, and thegenes were further amplified while introducing mutations. The presentinvention has been accomplished based on these findings.

[0021] Thus the present invention provides a molecule assigning agenotype to a phenotype, which comprises a nucleic acid portion having anucleotide sequence reflecting the genotype, and a protein portioncomprising a protein involved in exhibition of the phenotype, thenucleic acid portion and the protein portion being directly bound by achemical bond.

[0022] According to preferred embodiments of the present invention,there are provided the aforementioned assigning molecule wherein a3′-terminal end of the nucleic acid portion and a C-terminal end of theprotein portion are bound by a covalent bond, and the aforementionedassigning molecule wherein a 3′-terminal end of the nucleic acid portioncovalently bound to a C-terminal end of the protein portion ispuromycin.

[0023] According to another preferred embodiment of the presentinvention, there is also provided the aforementioned assigning moleculewherein the nucleic acid portion comprises a gene encoding a protein,and the protein portion is a translation product of the gene of thenucleic acid portion. The nucleic acid portion preferably comprises agene composed of RNA, and a suppressor tRNA bonded to the gene through aspacer. The suppressor tRNA preferably comprises an anticodoncorresponding to a termination codon of the gene. Alternatively, thenucleic acid portion may comprise a gene composed of RNA, and a spacerportion composed of DNA and RNA, or DNA and polyethylene glycol. Thenucleic acid portion may comprise a gene composed of DNA, and a spacerportion composed of DNA and RNA.

[0024] As further aspects of the present invention, there are provided amethod for constructing a molecule assigning a genotype to a phenotype,which comprises (a) boding a DNA comprising a sequence corresponding toa suppressor tRNA, to a 3′-terminal end of a DNA containing a genethrough a spacer, (b) transcribing the obtained DNA bonded product intoRNA, (c) bonding, to a 3′-terminal end of the obtained RNA, a nucleosideor a substance having a chemical structure analogous to that of anucleoside, which can be covalently bonded to an amino acid or asubstance having a chemical structure analogous to that of an aminoacid, and (d) performing protein synthesis in a cell-free proteinsynthesis system using the obtained bonded product as mRNA to bond anucleic acid portion containing the gene to a translation product of thegene; and a method for constructing a molecule assigning a genotype to aphenotype, which comprises (a) preparing a DNA containing a gene whichhas no termination codon, (b) transcribing the prepared DNA into RNA,(c) bonding a chimeric spacer composed of DNA and RNA to a 3′-terminalend of the obtained RNA, (d) bonding, to a 3′-terminal end of theobtained bonded product, a nucleoside or a substance having a chemicalstructure analogous to that of a nucleoside, which can be covalentlybonded to an amino acid or a substance having a chemical structureanalogous to that of an amino acid, and (e) performing protein synthesisin a cell-free protein synthesis system using the obtained bondedproduct as mRNA to bond a nucleic acid portion containing the gene to atranslation product of the gene.

[0025] According to a preferred embodiment of the present invention,there is provided the aforementioned construction method wherein thenucleoside or the substance having the chemical structure analogous tothat of the nucleoside is puromycin.

[0026] As another aspect of the present invention, there is provided amethod for constructing a molecule assigning a genotype to a phenotype,which comprises (a) preparing a DNA containing a gene which has notermination codon, (b) transcribing the prepared DNA into RNA, (c)bonding a chimeric spacer composed of DNA and polyethylene glycol to a3′-terminal end of the obtained RNA, (d) bonding, to a 3′-terminal endof the obtained bonded product, a nucleoside or a substance having achemical structure analogous to that of a nucleoside, which can becovalently bound to an amino acid or a substance having a chemicalstructure analogous to that of an amino acid, and (e) performing proteinsynthesis in a cell-free protein synthesis system using the obtainedbonded product as mRNA to bond a nucleic acid portion containing thegene to a translation product of the gene.

[0027] As another aspect of the present invention, there is provided amethod for constructing a molecule assigning a genotype to a phenotype,which comprises (a) preparing a DNA containing a gene which has notermination codon, (b) transcribing the prepared DNA into RNA, (c)bonding a spacer composed of double-stranded DNA to a 3′-terminal end ofthe obtained RNA, (d) bonding, to a 3′-terminal end of the obtainedbonded product, a nucleoside or a substance having a chemical structureanalogous to that of a nucleoside, which can be covalently bound to anamino acid or a substance having a chemical structure analogous to thatof an amino acid, and (e) performing protein synthesis in a cell-freeprotein synthesis system using the obtained bonded product as mRNA tobond a nucleic acid portion containing the gene to a translation productof the gene.

[0028] As a further aspect of the present invention, there is provided amethod for constructing a molecule assigning a genotype to a phenotype,which comprises (a) preparing a DNA containing a gene which has no atermination codon, and a nucleotide sequence of a spacer, (b)transcribing the prepared DNA into RNA, (c) bonding, to a 3′-terminalend of the obtained RNA, a nucleoside or a substance having a chemicalstructure analogous to that of a nucleoside, which can be covalentlybonded to an amino acid or a substance having a chemical structureanalogous to that of an amino acid, (d) adding a short chain PNA or DNAto a 3′-terminal end side portion of the gene in the obtained RNA bondedproduct to form a double-stranded chain, and (e) performing proteinsynthesis in a cell-free protein synthesis system using the obtainedbonded product as mRNA to bond a nucleic acid portion containing thegene to a translation product of the gene.

[0029] As a still further aspect of the present invention, there isprovided a method for protein evolution simulation, which comprises aconstruction step for constructing assigning molecules from a DNAcontaining a gene by any one of the construction methods mentionedabove, a selection step for selecting the assigning molecules obtainedin the construction step, a mutation introduction step for introducing amutation into a gene portion of an assigning molecule selected in theselection step, and an amplification step for amplifying the geneportion obtained in the mutation introduction step. In the method forevolution simulation, the construction step, the selection step, themutation introduction step and the amplification step are preferablyperformed repeatedly by providing the DNA obtained in the amplificationstep to the construction step. Further, there is provided an apparatusfor performing the aforementioned method for evolution simulation, whichcomprises a means for constructing assigning molecules, said meanscomprising a first bonding means for bonding a DNA comprising a sequencecorresponding to a suppressor tRNA to a 3′-terminal end of a DNAcontaining a gene through a spacer, a transcription means fortranscribing the DNA bonded product obtained by the first bonding meansinto RNA, a second bonding means for bonding, to a 3′-terminal end ofthe RNA obtained by a transcription means, a nucleoside or a substancehaving a chemical structure analogous to that of a nucleoside, which canbe covalently bound to an amino acid or a substance having a chemicalstructure analogous to that of an amino acid, and a third bonding meansfor performing protein synthesis in a cell-free protein synthesis systemusing the bonded product obtained by the second bonding means as mRNA tobond a nucleic acid portion containing the gene to a translation productof the gene, or a means for constructing assigning molecules, said meanscomprising a transcription means for transcribing a DNA containing agene into RNA, a first bonding means for bonding a chimeric spacercomposed of DNA and RNA, a chimeric spacer composed of DNA andpolyethylene glycol, a double-stranded spacer composed of DNA and DNA,or a double-stranded spacer composed of RNA and a short chain peptidenucleic acid (PNA) or DNA to a 3′-terminal end of the RNA obtained bythe transcription means, a second bonding means for bonding, to a3′-terminal end of the RNA-spacer bonded obtained by the first bondingmeans, a nucleoside or a substance having a chemical structure analogousto that of a nucleoside, which can be covalently bound to an amino acidor a substance having a chemical structure analogous to that of an aminoacid, and a third bonding means for performing protein synthesis in acell-free protein synthesis system using the bonded product obtained bythe second bonding means as mRNA to bond a nucleic acid portioncontaining the gene to a translation product of the gene; a selectionmeans for selecting the constructed assigning molecules; a mutationintroduction means for introducing a mutation into a gene portion of anassigning molecule selected; and an amplification means for amplifyingthe gene portion to which the mutation is introduced.

[0030] As a still further aspect of the present invention, there isprovided a method for assaying protein/protein or protein/nucleic acidintermolecular action, which comprises a construction step forconstructing assigning molecules by any one of the aforementionedconstruction methods, and an assay step for examining intermolecularaction of the assigning molecules obtained in the construction step withanother protein or nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031]FIG. 1 shows strategies for genotype (nucleic acid portion)assignment to phenotype (protein portion).

[0032]FIG. 2 shows a method for construction of the molecule assigningthe genotype to the phenotype of the present invention wherein a nucleicacid portion and a protein portion are bonded in a site-directed manner.

[0033]FIG. 3 shows chemically-modified portions of the 3′-terminal endsof nucleic acid portions, which are a point of the construction of themolecule assigning the genotype to the phenotype (in vitro virus).

[0034]FIG. 4 shows a method for construction of the molecule assigningthe genotype to the phenotype of the present invention wherein a nucleicacid portion and a protein portion are bonded in a non-site-directedmanner.

[0035]FIG. 5 is a photograph of electrophoresis image that shows spaceroptimization in the site-directed method. It shows the results of 4%polyacrylamide gel electrophoresis (in the presence of 8 M urea) of aDNA obtained through a process comprising translation of each RNA genomehaving a spacer in a length corresponding to each of the preparedfractions a, b, and c in the presence of a biotinylated lysyl tRNA in anE. coli cell-free translation system, specific absorption onstreptavidin-coated magnetic beads, reverse transcription, andamplification by PCR (staining was silver staining). Lane 1 is for thespacer length of fraction a (255-306 residues), Lane 2 is for the spacerlength of fraction 2 (102-238 residues), and Lane 3 is for the spacerlength of fraction c (0-85 residues).

[0036]FIG. 6 is a photograph of electrophoresis image showing bonding ofa nucleic acid portion and a protein portion in a site-directed method.The results were obtained by 18% polyacrylamide gel electrophoresis (inthe presence of 8 M urea and SDS): Lane 1 for a translation product ofmRNA encoding the 4 repeats region of a tau protein, which was obtainedin an E. coli cell-free translation system while labeled with[³⁵S]-methionine, and Lane 2 for a translation product of the mRNA whose3′-terminal end was bonded to sup tRNA having puromycin, and whose5′-terminal end was labeled with [³²P], which was obtained in an E. colicell-free translation system.

[0037]FIG. 7 is a photograph of electrophoresis image showing bonding ofnucleic acid portion and protein portion in the non-site-directedmethod. The results were obtained by 18% polyacrylamide gelelectrophoresis (in the presence of SDS): Lane 1 for a translationproduct of mRNA encoding the 4 repeats region of a tau protein, whichwas obtained in an E. coli cell-free translation system, while labeledwith [³⁵S]-methionine, and Lane 2 for a translation product of the mRNAwhose 3′-terminal end was bonded through a spacer to puromycin labeledwith [³²P] at the 5′ end, which was obtained in an E. coli cell-freetranslation system, and Lane 3 for the translation product of Lane 2digested with ribonuclease T2.

[0038]FIG. 8 shows an example of the method for constructing themolecule assigning the genotype to the phenotype (in vitro virus)according to the present invention.

[0039]FIG. 9 is a photograph of electrophoresis image showing bonding ofrCpPur to the C-terminal of the N-terminal half (1-165) of human tauprotein. Three kinds of genomes, i.e., one having a stop codon but not aDNA spacer (the first lane from the left), one having neither of a stopcodon and a DNA spacer (the second lane from the left), and one nothaving a stop codon but having a DNA spacer (the third lane from theleft) each at the 3′-terminal end of mRNA encoding the N-terminal half(1-165) of human tau protein, were constructed, and translated in acell-free translation system utilizing rabbit reticulocyte lysate in thepresence of rCpPur labeled with ³²P at 30° C. for 20 minutes. Thetranslation products were analyzed by 11.25% SDS-PAGE. The lane at theright end shows the result for a product obtained by translation of themRNA encoding the N-terminal half of human tau protein (1-165) in thepresence of [³⁵S]-methionine under the same condition mentioned above.

[0040]FIG. 10 is a photograph of electrophoresis image showinggeneration of in vitro viruses in a cell-free translation system. (A)shows a time course of generation of in vitro viruses. A genome composedof the mRNA encoding the N-terminal half of human tau protein (1-165), aDNA spacer (105 mer), a peptide acceptor, and rCpPur was translated in acell-free translation system utilizing rabbit reticulocyte lysate andcontaining [³⁵S]-methionine, and the translation product was examined ina time course (at 5 minutes, 10 minutes, 20 minutes, and 40 minutes) at30° C. The translation products were analyzed by 11.25% SDS-PAGE. Thefirst lane from the left shows the result obtained by using the RNAencoding the N-terminal half of human tau protein (1-165) as mRNA, andexamining incorporation of [³⁵S]-methionine into the protein under thesame condition as mentioned above. The lane at the right end shows theresult of in vitro virus genome labeled with ³²P. (B) shows influence ofconcentration of in vitro virus genome for the generation of in vitroviruses. Lane 1 shows the results for a genome labeled with [³²P]-rCpPurat the 3′-terminal end, Lane 2 for a genome (1.2 μg) to which rCpPur wasattached at its 3′-terminal end, Lane 3 for a genome (0.33 μg) to whichrCpPur was attached at its 3′-terminal end, and Lane 4 for a genome(0.64 μg) to which rCpPur was attached at its 3′-terminal end. As forLanes 2-4, the genomes were translated in a cell-free translation systemutilizing rabbit reticulocyte lysate and containing [³⁵S]-methionine at30° C. for 20 minutes. The translation products were analyzed by 11.25%SDS-PAGE.

[0041]FIG. 11 is a photograph of electrophoresis image showinggeneration of in vitro viruses in a cell-free translation system. An invitro virus genome composed of the mRNA encoding the N-terminal half(1-165) of human tau protein, a DNA spacer (105 mer), a peptideacceptor, and [³²P]-rCpPur was translated by utilizing rabbitreticulocyte lysate at 30° C. for 20 minutes. The translation productswere analyzed by 11.25% SDS-PAGE. The bonding of the genome and theprotein could be confirmed by digestion with mung bean nuclease. Whenthe translation product (Lane 3) was digested with mung bean nuclease,bands appeared (Lane 4) at the locations corresponding to monomer anddimer (Lane 1) of the N-terminal half of human tau protein (1-165). Lane2 shows the result for an in vitro virus genome labeled with ³²P.

[0042]FIG. 12 shows process steps of a protein evolution simulationmethod utilizing in vitro viruses.

BEST MODE FOR CARRYING OUT THE INVENTION

[0043] In this specification, some technical terms are used, and thosetechnical terms have the following meanings when herein used. The term“nucleic acid portion” means a bonded product of a nucleoside or asubstance having a chemical structure analogous to a nucleoside, forexample, RNA, DNA, PNA (peptide nucleic acid; polymers comprisingnucleic acids linked via amino acid analogues) and the like, and“protein portion” means a bonded product of an amino acid or a substancehaving a chemical structure analogous to an amino acid such asnaturally-occurring amino acids and non-naturally-occurring amino acids.The term “suppressor tRNA (sup tRNA)” means a tRNA which can suppressmutation by structural change, for example, reading a termination codonon mRNA as a codon corresponding to a certain amino acid. The expressionof “having a nucleotide sequence reflecting genotype” means to contain agene or a part thereof relating to a genotype. The expression of“containing a protein involved in exhibition of phenotype” means tocontain, for example, a protein whose expression itself is acharacteristic of phenotype, a protein involved in exhibition of acharacteristic of phenotype by its function as an enzyme or the like.

[0044] The spacer located at the 3′-terminal end side of the nucleicacid portion may be any spacer provided that it is a polymer substancepreferably having a length of not less than 100 Å, more preferably about100 to 1000 Å. Specifically, single-stranded chains of RNA or DNA,double-stranded chains of DNA and DNA, double-stranded chains of RNA andshort chain PNA or DNA (e.g., about 15 to 25 nucleotides), and polymermaterials such as polysaccharides, which are naturally-occurring orsynthetic, synthetic organic polymer substances such as polyethyleneglycols, preferably polyethylene glycols having a molecular weight ofabout 3,000 to 30,000 and the like can be mentioned.

[0045] The nucleic acid portion and the protein portion of the assigningmolecule of the present invention are linked through a chemical bondsuch as a covalent bond. In particular, preferred are those formed bybonding a nucleoside or a substance having a chemical structureanalogous to a nucleoside, or a linked product thereof present at the3′-terminal end of the nucleic acid portion to an amino acid or asubstance having a chemical structure analogous to an amino acid presentat the C-terminal end of the protein portion via a chemical bond, forexample, a covalent bond.

[0046] For the bonding between the nucleic acid portion and the proteinportion, for example, puromycin, 3′-N-aminoacylpuromycin aminonucleoside(PANS-amino acid), which have an amide bond as the chemical bond at the3′-terminal end of the nucleic acid portion, e.g., PANS-Gly wherein theamino acid portion is glycine, PANS-Val wherein the amino acid portionis valine, PANS-Ala wherein the amino acid portion is alanine, andfurther PANS-(any of the other amino acids) wherein the amino acidportion is an of the other amino acids, can be utilized.3′-N-Aminoacyladenosine aminonucleoside (AANS-amino acid), whichcomprises as the chemical bond an amide bond formed by dehydrationcondensation of the amino group of 3′-aminoadenosine and the carboxylgroup of an amino acid, for example, AANS-Gly wherein the amino acidportion is glycine, AANS-Val wherein the amino acid portion is valine,AANS-Ala wherein the amino acid portion is alanine, and furtherAANS-(any of the other amino acids) wherein the amino acid portion isany of the other amino acids, can also be utilized. Those composed of anucleoside or a nucleoside bound to an amino acid via an ester bond mayalso be used. Further, any other materials having a binding mode capableof binding a nucleoside or a substance having a chemical structureanalogous to a nucleoside and an amino acid or a substance having achemical structure analogous to an amino acid can also be utilized.

[0047] The molecule assigning the genotype to the phenotype of thepresent invention can be constructed by, for example, (1) a method wherethe binding of the nucleic acid portion and the protein portion isformed in a site-directed manner, or (2) a method where the bonding ofthe nucleic acid portion and the protein portion is formed in anon-site-directed manner, which will be explained hereinafter.

[0048] First, (1) the method where the bonding of the nucleic acidportion and the protein portion is formed in a site-directed manner willbe explained.

[0049] In this method, a molecule assigning a genotype to a phenotypecan be constructed by (a) bonding a DNA comprising a sequencecorresponding to sup tRNA, to the 3′-terminal end of a DNA containing agene through a spacer, (b) transcribing the obtained DNA bonded productinto RNA, (c) bonding, to the 3′-terminal end of the obtained RNA, anucleoside or a substance having a chemical structure analogous to thatof a nucleoside, which can be covalently bound to an amino acid or asubstance having a chemical structure analogous to that of an aminoacid, e.g., puromycin, (d) performing protein synthesis in a cell-freeprotein synthesis system, e.g., an E. coli cell-free protein synthesissystem, using the obtained bonded product as mRNA, and thus (e)affording a molecule assigning a genotype-to a phenotype comprising agene RNA (genotype) and a protein (phenotype) which is a translationproduct of the gene, which are chemically bound through a nucleoside ora substance having a chemical structure analogous to that of anucleoside, e.g., puromycin.

[0050] That is, according to this method of the present invention, whena termination codon comes into the A site of ribosome during the proteinsynthesis, a sup tRNA is correspondingly incorporated, and a nucleosideor a substance having a chemical structure analogous to that of anucleoside, e.g., puromycin, present at the 3′-terminal end of the suptRNA is bound to a protein by the action of peptidyl transferase (FIG.2). Therefore, this method is site-directed as for the formation of thebonding between the nucleic acid portion and the protein, which dependson the genetic code.

[0051] It has been known that puromycin (FIG. 3) inhibits the proteinsynthesis in bacteria (Nathans, D. (19.64) Proc. Natl. Acad. Sci. USA,51, 585-592; Takeda, Y. et al. (1960) J. Biochem. 48, 169-177) andanimal cells (Ferguson, J. J. (1962) Biochim. Biophys. Acta 57, 616-617;Nemeth, A. M. & de la Haba, G. L. (1962) J. Biol. Chem. 237, 1190-1193).Puromycin, whose structure resembles the structure of aminoacyl tRNA,reacts with peptidyl tRNA bound to the P site of ribosome, and it isreleased from ribosome as peptidyl puromycin, and thus interrupts theprotein synthesis (Harris, R. J. (1971) Biochim. Biophys. Acta 240,244-262).

[0052] It is not practical to purify native sup tRNA and bond it tomRNA, because of the problems concerning the purification of sup tRNAand the easily hydrolyzable ester bond at the 3′-terminal end of tRNA.Through investigations of tRNA identity, it has been elucidated thatunmodified tRNA may be aminoacylated like intact tRNA, and that theaminoacylated unmodified tRNA may be taken into ribosome, and translated(Shimizu, M. et al. (1992) J. Mol. Evol. 35, 436-443). The identity oftRNA is also utilized in order to prepare sup tRNA.

[0053] It has been reported that the aminoacyl synthetases of alanine,histidine, and leucine do not recognize the anticodons thereof (Tamura,K. et al. (1991) J. Mol. Recog. 4, 129-132). Therefore, it can beexpected that, by replacing the anticodon of tRNA for alanine with atermination codon (e.g., amber), tRNA for alanine (sup tRNA) would beincorporated corresponding to the termination codon.

[0054] In this respect, it comes into question whether tRNA whose5′-terminal-end side is not made up by RNAse P or the like, unlikeordinary tRNA, may enter into the A site of ribosome or not. This is themost important problem to be investigated in determining feasibility ofthe model of the present invention. It has been known that the3′-terminal ends of Brome Mosaic Virus (BMV) and Turnip Yellow MosaicVirus (TYMV) have a tRNA-like structure, and they are aminoacylated byaminoacyl synthetase, and incorporated at an efficiency of 1% in acell-free translation system (Chen, J. M. & Hall, T. C. (1973)Biochemistry 12, 4570-4574). Supposing that RNA of BMV is incorporatedeven by 1% by ribosome, it can be expected that RNA having intact tRNAat its 3′-terminal end may be incorporated more efficiently. Even if itis incorporated at an efficiency of 10% or less of that of intact tRNA,there is a reasonable possibility that it can win the competition withthe release factor by the concentration effect.

[0055] Therefore, before the experiment for bonding a protein to the3′-terminal end of mRNA-sup tRNA (mRNA ligated at its 3′-terminal endwith sup tRNA through a spacer), it was examined whether even sup tRNAseparated from mRNA entered into the A site of ribosome and was bound toa protein. A sup tRNA whose 3′-terminal end was bonded to puromycin wasactually prepared, and added to a cell-free protein synthesis system toexamine whether the sup tRNA portion entered into the A site of ribosomecorresponding to occurrence of a termination codon and bound to aprotein. The 4 repeats region of tau protein (127 residues) was used asmRNA (Goedert, M. (1989) EMBO J. 8, 392-399). As a result, when thetranslation was performed in a cell-free protein synthesis system, itcould be confirmed that the sup tRNA having puromycin at its 3′-terminalend was incorporated into the A site of ribosome corresponding to atermination codon and bound to a protein (FIG. 2).

[0056] Then, RNA-sup tRNA bonded products having different lengths ofthe spacer between mRNA and sup tRNA were constructed, and it wasattempted to select an optimum length of the spacer which afforded thebest efficiency of the incorporation of the sup tRNA portion into the Asite of ribosome by the in vitro selection method. As a result, it wasfound that the RNA-sup tRNA bonded product having a certain spacerlength was chemically bound to a protein that was a translation productthereof with a good efficiency.

[0057] In order to construct the molecule assigning the genotype to thephenotype of the present invention, a nucleoside or a substance having achemical structure analogous to that of a nucleoside, which is to bebonded to the 3′-terminal end of the nucleic acid portion, and can becovalently bound to an amino acid or a substance having a chemicalstructure analogous to that of an amino acid, e.g.,2′-deoxycytidylylpuromycin (dCpPur) and ribocytidylpuromycin (rCpPur)(FIG. 3), must be synthesized first.

[0058] An exemplary method for synthesizing dCpPur is as follows. First,puromycin-5′-monophosphate can be prepared by chemically phosphorylatingthe 5′-hydroxyl group of puromycin using phosphorus oxychloride andtrimethylphosphate. Then, the amino group of the amino acid portion andthe 2′-hydroxyl group of the ribose portion inpuromycin-5′-monophosphate can be protected by reactingpuromycin-5′-monophosphate with trifluoroacetic acid and trifluoroaceticanhydride. The protected product can be reacted with Bz-DMTdeoxycytidine in which the amino group of the pyrimidine ring and the5′-hydroxyl group of the ribose portion in deoxycytidine are protected,in the presence of a condensation agent, dicyclohexylcarbodiimide, andthen deprotected with acetic acid and ammonia to afford2′-deoxycytidylylpuromycin (dCpPur). pdCpPur can be obtained byphosphorylating the 5′-hydroxyl group of dCpPur with polynucleotidekinase.

[0059] The ribocytidylpuromycin can be prepared by condensing puromycinand rC-β-amidite having protective groups in the presence of tetrazole,and oxidizing and deprotecting the product.

[0060] Then, the construction of a bonded product constituting thenucleic acid portion for binding the nucleic acid portion and theprotein portion in a site-directed manner will be described hereinafter.

[0061] As the bonded product constituting the nucleic acid portion usedfor the site-directed method, for example, a bonded product comprising5′-(T7 promoter region)-(Shine-Dalgarno (SD) sequence region)-(mRNAregion)-(spacer region)-(sup tRNA region)-(puromycin region)-3′connected in this order in sequence can be mentioned.

[0062] In the construction of this bonded product for the nucleic acidportion, a plasmid comprising the 4 repeats region, which is amicrotuble-binding region of human tau protein called htau24 (Goedert,M. (1989) EMBO J. 8, 392-399), inserted downstream of T7 promoter(pAR3040) is constructed first, and it is digested with restrictionenzymes BglII and BamHI to afford a linear DNA. This DNA is used as atemplate, and amplification is carried out by PCR by using primers forupstream region containing T7 region (forward) and for downstream regioncontaining the SD region and a region around the initiation codon(backward), and Taq DNA polymerase.

[0063] In the above method, three methionines may be added to thebackward primer in order to enhance detection sensitivity forradioactive methionine in the protein portion after the proteinsynthesis. That is, leucine at position 4, and lysines at positions 5and 8 of the 4 repeats region are replaced with methionines. Eventually,the translated 4 repeats protein contains four methionines in total.Then, amplification is carried out by PCR using a DNA containing theaforementioned linearized 4 repeats region as a template, acomplementary chain of the backward primer mentioned above as a forwardprimer, and a backward primer which is designed so that the C-terminalend of the 4 repeats region should have an amber codon as thetermination codon.

[0064] The two kinds of DNA fragments amplified by the PCR, namely, theDNA fragment containing the T7 promoter and the SD region and the DNAfragment containing the 4 repeats region are mixed, initially extendedwithout primers, and then amplified by PCR again by using a primercontaining the sequence of T7 promoter as the forward primer, and aprimer containing a termination codon at the C-terminal of the 4 repeatsregion as the backward primer.

[0065] This DNA bonded product (T7 promoter-SD-4 repeats) is ligated toa double-stranded DNA fragment having cohesive ends at the both ends andcomposed of 17 residues in tandem by using DNA ligase to afford ligationproducts having different spacer lengths.

[0066] After the ligation, the product was fractionated into threefractions (a, b, c) based on the length by polyacrylamide gelelectrophoresis (PAGE). The spacer is represented as (17)n wherein n=15to 18 for the fraction a, n=6 to 14 for the fraction b, and n=0 to 5 forthe fraction c. As the sup tRNA, a native alanyl tRNA whose severalsites and anticodon are modified into amber (UAG) is prepared bychemical synthesis. This sup tRNA is ligated to the ligation products ofthe fractions a, b and c having different spacer lengths by using T4 DNAligase. For the ligation site, an excessive amount of a single-strandedbacking DNA is used, and after once melted by temperature elevation, thestrands are annealed, and a complementary strand is formed, and ligated.After the ligation, the ligation product is amplified by PCR usingprimers for the 5′ end and 3′-terminal end of the ligation product. ThisDNA ligation product is transcribed by using T7 RNA polymerase to forman RNA ligation product.

[0067] By ligating the pdCpPur chemically synthesized in the above tothe 3′-terminal end of this RNA ligation product using T4 RNA ligase,there can be obtained an RNA ligation product, 5′-(T7 promoterregion)-(SD region)-(4 repeats region)-(spacer region)-(sup tRNAregion)-(puromycin)-3′, which can be used as a gene in a cell-freeprotein synthesis system.

[0068] The protein synthesis is performed by adding the above RNAligation product as mRNA to a cell-free protein synthesis system such ascell-free protein synthesis extracts of E. coli or rabbit reticulocytes.In order to obtain an optimum spacer length for obtaining the mostefficient bonding of the nucleic acid portion (RNA) and the proteinportion, the following experiment is performed.

[0069] That is, the protein synthesis is performed in a cell-freeprotein synthesis system by using aforementioned RNA ligation productshaving three kinds of different spacer lengths, corresponding to thefractions a, b and c, as the gene. In this synthesis, by adding tRNAcharged with modified lysine comprising biotin bound through the ε-aminogroup of the lysine, biotinyllysine is incorporated in several positionsof lysine residues in the translated 4 repeats protein. After theprotein synthesis, magnetic beads coated with streptavidin on theirsurfaces are added to isolate the protein incorporating the biotin.

[0070] If the nucleic acid portion (RNA) has bonded to the proteinportion through puromycin, the nucleic acid portion (RNA) should bebound to the C-terminal end of the protein. When reverse transcriptionwas performed by using a sequence corresponding to the N-terminal regionof the 4 repeats as a forward primer and the 3-terminal end portion ofsup tRNA as a backward primer and analyzed by polyacrylamide gelelectrophoresis to confirm whether the RNA-protein-bonded product wasactually picked up by the magnetic beads, a band of reverse transcribedDNA was observed only for the spacer length of the fraction c. Thismeans that the RNA ligation product having the spacer length of thefraction c is most efficiently bound to the protein portion.

[0071] Now, it will be explained about (2) the method where the bondingof the nucleic acid portion and the protein portion is formed in anon-site-directed manner.

[0072] In this method, a molecule assigning a genotype to a phenotypecan be constructed by (a) preparing a DNA containing a gene which has notermination codon, (b) transcribing the prepared DNA into RNA, (c)bonding a chimeric spacer composed of DNA and RNA to the 3′-terminal endof the obtained RNA, (d) bonding, to the 3′-terminal end of the bondedproduct, a nucleoside or a substance having a chemical structureanalogous to that of a nucleoside, which can be covalently bound to anamino acid or a substance having a chemical structure analogous to thatof an amino acid, e.g., puromycin, (e) performing protein synthesis in acell-free protein synthesis system using the obtained bonded product asmRNA, and thus (f) affording a molecule assigning a genotype to aphenotype comprising a gene RNA and a protein which is a translationproduct of the gene, which are chemically bound through puromycin or thelike.

[0073] That is, according to this method of the present invention, anucleoside or a substance having a chemical structure analogous to thatof a nucleoside, e.g., puromycin, present at the 3′-terminal end of thenucleic acid portion does not enter into the A site of ribosomecorresponding to the termination codon of mRNA on ribosome, but randomlyenters depending on the spacer length, and puromycin or the like at the3′-terminal end of the RNA-DNA chimera nucleic acid portion ischemically bound to a protein by the action of peptidyl transferase(FIG. 4). Therefore, this method is non-site-directed as for theformation of the bonding between the nucleic acid portion and theprotein, which does not depend on the genetic code.

[0074] In this method, the molecule assigning the genotype to thephenotype can be constructed by using a non-site-directed ligationproduct for the nucleic acid portion in the same manner as in theaforementioned site-directed method of (1).

[0075] As the ligation product constituting the nucleic acid portionused for the non-site-directed method, for example, a ligation productcomposed of 5′-(T7 promoter region)-(Shine-Dalgarno (SD) sequenceregion)-(mRNA region)-(spacer region)-(puromycin region)-3′ connected inthis order in sequence can be mentioned.

[0076] In the construction of this ligation product for the nucleic acidportion, the construction from the T7 promoter region to the end of the4 repeats region may be similar to that explained for the constructionof the ligation product for the nucleic acid portion used in (1) thesite directed method mentioned above, provided that a primer designednot to have a termination codon by replacing the two termination codonsat the C-terminal end of the 4 repeats, ochre (CTG) and amber (TAA),with CAG (glutamine) and AAA (lysine), respectively, is used as abackward primer for the PCR amplification of the ligation productconstructed above used as a template.

[0077] This DNA ligation product is transcribed as a template by usingT7 RNA polymerase to afford a corresponding RNA ligation product. Thissingle-stranded RNA ligation product is separately ligated to each ofsingle-stranded chemically-synthesized DNA linkers (chain length; 20,40, 60, and 80 nucleotides) by using T4 RNA ligase. Then, each ligationproduct is ligated to a single-stranded DNA-RNA chimeric oligonucleotidecomprising 25 residues (DNA; 21 residues, RNA; 4 residues), which isdesignated as peptide acceptor, by using T4 DNA ligase in the presenceof a single-stranded backing DNA.

[0078] Because the sequence of the peptide acceptor contains the3′-terminal end sequence of alanyl tRNA, and it enhances theincorporation of a puromycin derivative into the A site of ribosome, itis preferable to use the peptide acceptor between the spacer region andthe puromycin region.

[0079] By ligating the pdCpPur chemically synthesized in the above tothe 3′-terminal end of the above ligation product using T4 RNA ligase,there can be obtained an RNA-DNA chimeric ligation product, 5′-(T7promoter region (RNA))-(SD region (RNA))-(4 repeats region(RNA))-(spacer region (DNA))-(peptide acceptor region)-(puromycin)-3′,which can be used as a gene for a cell-free protein synthesis system.

[0080] If protein synthesis is performed by using the RNA-DNA chimericligation product mentioned above as a gene in a cell-free proteinsynthesis system, there can be obtained a bonded product comprising anucleic acid portion (RNA-DNA chimeric ligation product, genotype) and aprotein portion (phenotype), which are connected by a chemical bondthrough puromycin.

[0081] In the aforementioned method, a chimeric spacer of DNA andpolyethylene glycol can also be used instead of the chimeric spacer ofDNA and RNA.

[0082] In the above method, a spacer composed of a double-stranded chainof DNA and DNA, or a double-stranded chain of RNA and short chain PNA orDNA (e.g., about 15 to 25 nucleotides) may also be used instead of thechimeric spacer of DNA and RNA. The spacer composed of the double strandof DNA and DNA is not necessary to be double-stranded in its fulllength, and it may be double-stranded for most part (usually, severalresidues at the both ends are single-stranded, and remaining portion isdouble-stranded). The double-stranded spacer composed of RNA and shortchain PNA or DNA can also be prepared by (a) preparing a DNA containinga gene which has no termination codon, and a nucleotide sequence of aspacer, (b) transcribing the prepared DNA into RNA, (c) bonding, to the3′-terminal end of the obtained RNA, a nucleoside or a substance havinga chemical structure analogous to that of a nucleoside, which can becovalently bound to an amino acid or a substance having a chemicalstructure analogous to that of an amino acid, and (d) adding a shortchain PNA or DNA to a 3′-terminal-end side portion of the gene in theobtained RNA bonded product to form a double-stranded chain.

[0083] The genetic engineering techniques mentioned in the presentspecification such as isolation and preparation of nucleic acids,ligation of nucleic acids, synthesis of nucleic acids, PCR, constructionof plasmids, and translation in cell-free system can be performed by themethods described in Sambrook et al. (1989) Molecular Cloning, 2ndEdition, Cold Spring Harbor Laboratory Press, or similar methods unlessotherwise indicated.

[0084] The assigning molecule of the present invention can also beobtained by successively bonding each of the elements by any knownchemical bonding methods in addition to the methods exemplified above.

[0085] The protein evolution simulation method of the present inventionis a method comprising steps of (1) construction of in vitro virusgenomes, (2) completion of in vitro viruses, (3) selection process, (4)introduction of mutation, and (5) amplification, as shown in FIG. 12.These steps or repetition of these steps as required allows modificationand creation of functional proteins. Among these steps, the steps of (1)and (2) can be performed by the construction methods explained above indetail. That is, the step (1) corresponds to construction of the bondedproduct comprising the nucleoside or the substance having the chemicalstructure analogous to that of the nucleoside, and the step (2)corresponds to the construction of the assigning molecule from thebonded product. The steps of (3), (4) and (5) will be describedhereinafter.

[0086] The selection process of (3) means a process of evaluatingfunction (biological activity) of protein portions constituting the invitro viruses, and selecting in vitro viruses based on a desiredbiological activity. Such a process has been known, and described in,for example, Scott, J. K. & Smith, G. P. (1990) Science, 249, 386-390;Devlin, P. E. et al. (1990) Science, 249, 404-406; Mattheakis, L. C. etal. (1994) Proc. Natl. Acad. Sci. USA, 91, 9022-9026 and the like.

[0087] Then, mutations are introduced into the nucleic acid portions ofthe selected in vitro viruses, and the in vitro viruses are amplified byPCR or the like in the steps of (4) introduction of mutation, and (5)amplification. When the nucleic acid portion of in vitro viruses iscomposed of RNA, mutation can be introduced after a cDNA is synthesizedby reverse transcriptase. The amplification of the nucleic acid portionmay also be performed while introducing mutation. The introduction ofmutation can be readily performed by already established error-prone PCR(Leung, D. W., et al., (1989) J. Methods Cell Mol. Biol., 1, 11-15),Sexual PCR (Stemmer, W. P. C. (1994) Proc. Natl. Acad. Sci. USA 91,10747-10751) or the like.

[0088] (1) In vitro virus genomes can be constructed by using nucleicacid portions for in vitro viruses which have been introduced withmutation and amplified, (2) in vitro viruses can be completed by usingthe in vitro virus genomes, (3) in vitro viruses can be selected basedon a desired biological activity, and (4) mutation introduction andamplification can be carried out. By repeating these steps as required,modification and creation of functional proteins can be realized.

[0089] The means contained in the apparatus of the present invention forperforming the aforementioned protein evolution simulation methodthemselves are known ones, and operations in these means such asaddition of reagents, stirring, temperature control, and evaluation ofbiological activity can be performed according to the methods known perse. By combining these operations, an automatic or semi-automaticapparatus of the present invention can be constructed.

[0090] The step of constructing assigning molecules in the method forassaying protein/protein or protein/nucleic acid intermolecular actionof the present invention generally comprises steps of (1) synthesizingmRNA from a gene library or a cDNA library, and constructing an in vitrogenome, and (2) constructing an in vitro virus comprising mRNA and acorresponding protein, which are bonded on ribosome, by utilizing acell-free protein synthesis system.

[0091] The step (1) corresponds to synthesis of mRNA using RNApolymerase from cDNA of DNA of which sequence has been known and whichcontains a sequence corresponding to ORF, cDNA of DNA of which sequenceis unknown and which contains a fragment resulting from fragmentationwith a suitable restriction enzyme or the like, and construction of anin vitro virus genome by utilizing the mRNA.

[0092] The steps of above (1) in vitro virus genome construction, and(2) in vitro virus construction can be performed by the constructionmethods explained above in detail.

[0093] The assay step for examining intermolecular action betweenassigning molecules and other proteins or nucleic acids (DNA or RNA)usually comprises steps of (3) selecting only proteins having aparticular function from the in vitro viruses constructed in the step(2), and (4) subjecting selected in vitro viruses to reversetranscription, amplification, and sequencing.

[0094] In the step (3), target proteins or nucleic acids (DNA or RNA)and other substances, for example, saccharides and lipids, are bound toa microplate, beads or the like beforehand through covalent bonds ornon-covalent bonds, and the in vitro viruses constructed in the step (2)are added thereto, to react under a certain temperature condition for acertain period of time, and it is washed to remove in vitro viruseswhich has not been bound to the target. Then, the in vitro viruses whichhave been bound to the target are released. This step can be performedby the already-established ELISA (Enzyme Linked Immunosorbent Assay,Crowther, J. R. (1995) Methods in Molecular Biology, Vol. 42, HumanaPress Inc.) or a similar technique.

[0095] In the step (4), the in vitro viruses released in the step (3)are reverse-transcribed and amplified by reverse transcription PCR, andthe amplified DNA was sequenced directly or after cloning.

[0096] According to the assay method of the present invention, itbecomes possible to identify a function of a gene product (protein)corresponding to a gene whose function is unknown by (1) synthesizingmRNA from gene DNA whose sequence is known or unknown to construct invitro virus genomes, (2) constructing in vitro viruses by using the invitro virus genomes, (3) selecting only those binding to a targetprotein or nucleic acid or other substances, for example, saccharidesand lipids from among the in vitro viruses, and (4) subjecting selectedin vitro viruses to reverse transcription, amplification, cloning andsequencing.

[0097] To perform the method for assaying intermolecular actionmentioned above, an apparatus can be constructed, by combining knownappropriate means. The means contained in the apparatus may be per seknown ones, and operations in these means such as addition of reagents,stirring, temperature control, and evaluation of biological activity canbe performed according to the methods known per se. By combining theseoperations, an automatic or semi-automatic apparatus for assayingintermolecular action can be constructed.

EXAMPLES

[0098] The present invention will be more specifically explained withreference to the following examples. However, the following examplesshould be construed to be an aid for obtaining more specificunderstanding of the present invention, and the scope of the presentinvention is not no way limited by these examples.

Example 1

[0099] Preparation of in vitro Virus (1)

[0100] <1> Preparation of 3′-terminal-end Portion of Nucleic AcidPortion

[0101] (a) Synthesis of Phosphorylated Puromycin (pPur)

[0102] Materials:

[0103] Puromycin(3′-[α-amino-p-methoxyhydro-cinnamamido]-3′-deoxy-N,N′-dimethyl-adenosine)was purchased from Sigma. Phosphorus oxychloride, and trimethylphosphate were purchased from Wako Pure Chemicals.

[0104] Methods:

[0105] A solution formed by mixing phosphorus oxychloride (1.5 mmol) andtrimethyl phosphate (11.4 mmol) was ice-cooled, and puromycin (0.3 mmol)was added thereto to mix sufficiently, and the mixture was allowed toreact at 0° C. for 7 hours (Yoshikawa, M. et al. (1969) Bull. Chem. Soc.Jap. 42, 3505-3508). The reaction mixture was then added to anice-cooled mixture of acetone (40 ml) and ether (20 ml) containingsodium perchlorate (NaClO₄, 0.4 g), and stirred sufficiently. Then,water (720 ml) was added to the mixture, and the mixture was stirred at4° C. for 24 hours to hydrolyze the chlorine group. The productprecipitated after the hydrolysis was separated by centrifugation, andwashed with acetone and ether. The resulting white powder was dried invacuo to afford phosphorylated puromycin with a yield of 70 to 90% basedon the puromycin.

[0106] (b) Protection of Phosphorylated Puromycin by Acetylation

[0107] Materials:

[0108] Trifluoroacetic acid (TFA) was purchased from Nacalai Tesque.Trifluoroacetic anhydride (TFAA) was purchased from Wako Pure Chemicals.

[0109] Methods:

[0110] The dried phosphorylated puromycin (0.2 mmol) and TFA (5 ml) weremixed, and TFAA (2 ml) was added to the mixture at −10° C., followed bystirring. The mixture was allowed to react at room temperature for 1hour with stirring (Weygand, F. & Gieger, R. (1956) Chem. Ber. 89,647-652). The reaction was quenched by adding water (50 ml), and the TFAwas removed by repeating a procedure comprising addition of water (10ml) and evaporation to dryness under reduced pressure 5 times. Finally,water (50 ml) was added to the resulting product, followed bylyophilization to afford phosphorylated puromycin in which the aminogroup of the amino acid portion in puromycin and the 2′-hydroxyl groupof the ribose portion were protected with acetyl groups with a yield of50 to 60% based on the phosphorylated puromycin.

[0111] (c) Synthesis of dCpPur (2′-deoxycytidyl(3′→5′)puromycin)

[0112] Materials:

[0113] BZ-DMT deoxycytidine(N⁴-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine) was purchasedfrom Sigma, and DCC (dicyclohexyl carbodiimide) was purchased fromWatanabe Chemical. The pyridine was purchased from Nacalai Tesque.

[0114] Methods:

[0115] The phosphorylated puromycin protected with acetyl groups (40μmol) and BZ-DMT deoxycytidine (600 μmol) were dehydrated by repeating aprocedure comprising addition of pyridine (2 ml) and evaporation todryness three times, and finally pyridine (2 ml) was added thereto. DCC(400 μmol) was added to the mixture with stirring, and the mixture wasallowed to react at room temperature for 3 days to 2 weeks (Ralph, R. K.et al. (1965) J. Am. Chem. Soc. 87, 5661-5670; and Harris, R. J. et al.(1972) Can. J. Biochem. 50, 918-926). After the reaction, the DMT groupwas removed by a reaction with 80% acetic acid (5 ml) for two hours.Then, the acetyl group was removed by a reaction with concentratedaqueous ammonia/ethanol (6 ml, volume ratio: 2:1) at 20° C. for 2 days.The concentrated aqueous ammonia was removed by evaporation underreduced pressure, and the residue was dissolved in water (40 ml). Theresulting solution was applied on a column packed with QAE-Sephadex A-25(Pharmacia) and adsorbed thereon. Fractions containing the targetproduct were eluted with 0.5 M triethylamine carbonate (TEAB, pH 7.5),lyophilized, and finally separated by HPLC to afford deprotected dCpPurwith a yield of 1 to 5% based on the puromycin.

[0116] <2> Preparation of Nucleic Acid Portion (in vitro Virus Genome)

[0117] Two kinds of in vitro virus genomes, i.e., (1) one for bonding anucleic acid portion and a protein portion in a site-directed manner,and (2) one for bonding a nucleic acid portion and a protein portion ina non-site-directed manner, were prepared.

[0118] Materials:

[0119] An E. coli cell-free protein synthesis system (E. coli S30Extract System for Linear Templates) was purchased from Promega. T7 RNApolymerase, T4 DNA ligase, T4 DNA kinase, human placenta ribonucleaseinhibitor, EcoRI, BamHI, and deoxyribonucleotides were purchased fromTakara Shuzo. Restriction enzymes BstNI and BglII were purchased fromNew England Labs. As for [³⁵S]-methionine, and [γ-³²P]-ATP, those fromAmersham, and as for Taq DNA polymerase, those from Kurabo and Grainerwere used. As for the other biochemical reagents, those from Sigma andWako Pure Chemicals were used. A plasmid containing themicrotuble-binding region of human tau protein (4 repeats) (pAR3040) wasprepared by picking up the full length gene of human tau protein from acDNA library of human brain cloned in λZAPII by PCR, introducing thegene into a plasmid, amplifying only the 4 repeats region in theplasmid, and introducing the amplified product into a plasmid. As thePCR (polymerase chain reaction) apparatuses, Model PTC-100 (MJ Research)and Model ASTEC PC800 (Astec) were used.

[0120] (1) Preparation of Genome for Site-directed Bonding

[0121] A. Preparation of DNA for Mutated 4 Repeats Portion

[0122] 1) A plasmid (pAR3040) comprising microtuble region (4 repeats)of human tau protein (Goedert, M. (1989) EMBO J. 8, 392-399) wasconstructed, and linearized by digestion with restriction enzymes BglIIand BamHI.

[0123] 2) The 4 repeats portion containing the T7 promoter region andthe Shine-Dalgarno sequence was amplified by PCR from the genomeprepared above. For this amplification, as primers, Left+ (SEQ ID NO: 1)was used for 5′ side, and Right− (SEQ ID NO: 2) for the 3′ side. Right−had such a sequence that the leucine before the ochre termination codonshould be mutated into amber termination codon. The PCR conditions were92° C./30 seconds for denaturation, 65° C./30 seconds for annealing, and73° C./1 minute for elongation, and this cycle was repeated 30 times.

[0124] 3) Then, the amplified genome was purified, and mutated byutilizing PCR in order to promote the incorporation of methionine andhence enhance detection of radioactive isotope. That is, primers Left−(SEQ ID NO: 3), and Right+ (SEQ ID NO: 4) containing a region desired tobe mutated were synthesized. First, using the DNA of the above 2) as atemplate, it was amplified by PCR with primers Left+ and Left−, and theamplified DNA was designated as “Left”. Amplification by PCR was alsoperformed with primers Right+, and Right−, and the amplified DNA wasdesignated as “Right”. After 5% polyacrylamide denatured gelelectrophoresis, “Left” and “Right” were excised form the gel, andextracted. The excised Left and Right were first amplified by PCR underthe same conditions as mentioned above without primers. Further, 1 μltaken from this reaction mixture was used as a template, and it wasamplified by PCR under the same conditions with primers Left+ andRight−. From the above procedure, DNA of the mutated 4 repeats portionin which the number of methionine was increased from one to four wasprepared.

[0125] B. Ligation of Alanine Suppressor tRNA (Ala-sup tRNA), ContainingSpacers Having Different Lengths to 4 Repeats Portion

[0126] 1) The BamH1 site located in the 3′-terminal-end sequence of theabove 4 repeats portion obtained in the above A was digested with BamH1.Then, to remove 3′-terminal-end fragment at the BamH1 site, only the 4repeats portion in 5′-terminal-end sequence was extracted and purifiedby using QIAquick PCR Purification Kit (QIAGEN).

[0127] 2) The purified product of the above 1), and Spacer-A (SEQ ID NO:5) which was phosphorylated at 5′-terminal end by T4 kinase were ligatedby using T4 DNA ligase while they were backed with Spacer-B (SEQ ID NO:6).

[0128] 3) Spacer-C (SEQ ID NO: 7) which was phosphorylated by T4 kinase,and Spacer-B which had a region complementary to Spacer-C were ligatedby using T4 DNA ligase. The reaction was performed at 15° C. for 2hours. Then, the product was purified by ethanol precipitation.

[0129] 4) The products of the above sections 2) and 3), Spacer-D (SEQ IDNO: 8), and sup tRNA (SEQ ID NO: 9) which was phosphorylated at5′-terminal end were dissolved in T4 DNA ligase buffer, denatured at 85°C. for 2 minutes, and cooled on ice. After addition of T4 DNA ligase, itwas allowed to react at 15° C. for 2 hours, and subjected to phenolextraction, and ethanol precipitation.

[0130] 5) The product obtained in the above 4) was used as a template,and amplification was carried out by PCR using the primer Left+, and aprimer 3′Pur- (SEQ ID NO: 10) under the conditions of 92° C./30 secondsfor denaturation, 65° C./30 seconds for annealing, and 73° C./1 minutefor elongation, which cycle was repeated 30 times. The product wassubjected to polyacrylamide denatured gel electrophoresis. Three regionsA, B and C exhibiting different migration distances were excised, andDNA was extracted from the regions.

[0131] 6) The DNAs extracted from A, B and C in the above 5) and havingdifferent lengths were used as templates and amplification were againcarried out by PCR under the same conditions, and lengths of theproducts were determined by electrophoresis, and they were used astemplate DNA for transcription. As a result, it was found that thenumbers of Spacer-C inserted into each product of the fractions were 0-5for the fraction c, 6-14 for the fraction b, and 15-18 for the fractiona.

[0132] C. Preparation of RNA Genome and Ligation of dCpPur

[0133] The regions A, B and C obtained in the above B was transcribedinto RNA at 37° for 2 hours by using T7 polymerase. Further, the dCpPurobtained in the above <1> Preparation of 3′-terminal-end portion of thenucleic acid portion was phosphorylated in the presence of ATP by usingT4 polynucleotide kinase at 15° C. for 24 hours, and ligated to theaforementioned transcribed RNA genome by using T4 RNA ligase at 4° C.for 50 hours. From this procedure, an RNA genome comprising sup tRNAhaving puromycin at its 3′-terminal end could be constructed.,

[0134] (2) Preparation of Genome for Non-site-directed Bonding

[0135] A. Preparation of DNA and RNA for Mutated 4 Repeats Portion

[0136] DNA of the mutated 4 repeats portion was prepared principally thesame method as the aforementioned (1) A. However, the termination codonswere eliminated by changing the two termination codons, amber and ochre,to glutamine and lysine, respectively, and a new primer New/Right− (SEQID NO: 10) was synthesized in order to make the 3′-terminal-end sequencepurine-rich, and used with Left+ for PCR amplification. Theamplification by PCR was performed under the conditions of 92° C./30seconds for denaturation, 65° C./30 seconds for annealing, and 73° C./1minute for elongation, which cycle was repeated 30 times. This DNA wasused as a template to obtain an RNA genome through a reaction at 37° C.for 2 hours utilizing T7 polymerase.

[0137] B. Ligation of Spacers 1 to 4

[0138] After a reaction at 36° C. for 1 hour with T4 polynucleotidekinase, of Spacer 1 which was a DNA composed of 21 nucleotides (SEQ IDNO: 11), Spacer 2 which was a DNA composed of 40 nucleotides (SEQ ID NO:12), Spacer 3 which was a DNA composed of 60 nucleotides (SEQ ID NO:13), or Spacer 4 which was a DNA composed of 80 nucleotides (SEQ ID NO:14), the RNA obtained in the above A was ligated to the spacers througha reaction at 10° C. for 48 hours using T4 RNA ligase.

[0139] C. Ligation of Peptide Acceptor (P-Acceptor)

[0140] A peptide acceptor (P-Acceptor, SEQ ID NO: 15), which was achimeric nucleic acid composed of 21-nucleotide DNA and 4-nucleotideRNA, i.e., 25 nucleotides in total, was synthesized in order to enhancethe incorporation efficiency into ribosomes by ligating it at its3′-terminal end to dCpPur. To phosphorylate the 5′-terminal end ofP-Acceptor, it was reacted at 36° C. for 1 hour using T4 polynucleotidekinase. Then, the product was backed with Back3′ (SEQ ID NO: 16) havinga complementary sequence thereto, and ligated to the 3′-terminal end ofeach of the spacers prepared in the aforementioned B through a reactionat 16° C. for 2 hours using T4 RNA ligase. This P-Acceptor was alsodirectly ligated to the 3′-terminal end of the RNA obtained in the aboveA through a reaction at 10° C. for 48 hours using T4 RNA ligase, and theproduct was designated as Non-Spacer genome.

[0141] D. Ligation of dCpPur

[0142] The dCpPur obtained in the above <1> Preparation of3′-terminal-end portion of nucleic acid portion was phosphorylated byusing T4 polynucleotide kinase at 15° C. for 24 hours, and ligated tothe 3′-terminal end of each of the genomes prepared in the above C byusing T4 RNA ligase at 4° C. for 50 hours. By this procedure, chimericRNA genomes comprising puromycin at its 3′-terminal end could beconstructed.

[0143] <3> Optimization of Nucleic Acid Portion

[0144] A. Site-directed Method

[0145] Each of the RNA genomes prepared in the above <2>, (1), whichwere classified into each of the lengths corresponding to the fractionsa, b and c, was translated in 50 μl of E. coli cell-free translationsystem [E. coli S30 Extract Systems for Linear Templates (Promega)]containing biotinylated lysyl tRNA (Promega), and after addition of 5 mgof streptavidin coated magnetic beads (Dynabeads, Dynal) to eachreaction tube, it was incubated at room temperature for 1 hour. Then,the Dynabeads were collected by a magnet, and the supernatant wasaspirated. The remained Dynabeads were washed 2 times with B&W buffer(1000 μl). The beads were further washed twice with RT-PCR buffer (500μl), and resuspended in RT-PCR buffer (500 μl). The suspension (50 μl)was transferred into a 500 μl Eppendorf tube, and after the Dynabeadswere immobilized with a magnet, the supernatant was aspirated. To theremained Dynabeads, RT-PCR buffer, reverse transcriptase, and Taqpolymerase [Access RT-PCR System (Promega)] were added. Reversetranscription was performed at 48° C. for 1 hours, and PCR was performedunder the conditions of 94° C./30 seconds, 65° C./40 seconds, and 68°C./1 minute and 40 seconds, which cycle was repeated 40 times, usingprimers of Right+ (SEQ ID NO: 4) and 3′Pur- (SEQ ID NO: 10). The resultsof the analysis of the fractions a, b and c by electrophoresis wereshown is FIG. 5.

[0146] A band was detected from the group of the fraction c (Lane 3 inFIG. 5). This band was separated from the gel by electrophoresis,ligated to “Left” having the T7 promoter and the Shine-Dalgarno regionby PCR, and amplified by PCR using the primers Left+ (SEQ ID NO: 3) and3′Pur- (SEQ ID NO: 10). This genome was designated as “Stranger”.

[0147] Then, to examine whether the protein actually translated fromStranger was bonded to the mRNA portion (RNA genome portion), aftertranscription, it was ligated with pdCpPur at its 3′-terminal end usingT4 RNA ligase, dephosphorylated at 5′-terminal end of RNA using HKphosphatase (Epicentre) at 30° C. for 1 hour, and labeled with[γ-³²P]-ATP using T4 polynucleotide kinase. The product was added to anE. coli cell-free translation system as mRNA, and allowed to react at37° C. for 1 hour and 40 minutes. The results of 18% SDS-PAGE of theproduct are shown in FIG. 6. From the results, it can be seen that thenucleic acid portion (genotype) and the protein portion (phenotype) werebonded to form in vitro viruses, i.e., molecules that assign a genotypeto a phenotype, at a rate of about 80% or more.

[0148] B. Non-site-directed Method

[0149] Because a short spacer was already used in the site-directedmethod, dCpPur, which had been phosphorylated at 5′-terminal end in thepresence of [γ-³²P]-ATP using T4 polynucleotide kinase, was ligated tothe 3′-terminal end of “Non-spacer” RNA genome without a spacer througha reaction using T4 RNA ligase at 4° C. for 50 hours. The product wasadded to an E. coli cell-free translation system together with mRNAencoding the ordinary 4 repeats, and allowed to react at 37° C. for 1hour and 30 minutes. This reaction mixture (10 μl) digested withribonuclease T2, and an equal amount of the reaction mixture wereelectrophoresed by 18% SDS-PAGE, and analyzed by an image analyzerBAS2000 (Fujifilm) (FIG. 7).

[0150] As a result, as for the sample digested with ribonuclease T2, aband appeared at the same migration distance as that of the control, the4 repeats labeled with [³⁵S]-methionine, because the sample containedthe released protein portion. On the other hand, as for the sample nottreated, a band appeared above the 4 repeats protein, i.e., it was foundthat the band reflected a clearly larger molecular weight. This band didnot correspond to the labeled mRNA itself (about 400 nucleotides),because it migrated a longer distance than the tRNA. Therefore, it wasidentified as a substance composed of bonded RNA and protein. That is,these results demonstrated that the nucleic acid portion was bonded tothe protein portion in a non-site-directed manner.

Example 2

[0151] Preparation of in vitro Virus (2)

[0152] <1> Preparation of 3′-terminal-end Portion of Nucleic AcidPortion 4

[0153] (a) Synthesis of rCpPur (ribocytidyl(3′→5′)puromycin

[0154] Materials:

[0155] Each material was purchased from the following manufacturers:Puromycin from Sigma, rC-β-amidite(N⁴-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-tert-butyldimethylsilyl)-cytidine-3′-O-[O-(2-cyanoethyl)-N,N′-diisopropyl-phosphoramidite])from Japan PerSeptive, tetrazole from Japan Millipore,tetrabutylammonium fluoride from Aldrich, QAE-Sephadex from Pharmacia,and silica gel for chromatography from Merck.

[0156] Methods:

[0157] Puromycin (50 mg, 92 μmol) was dissolved in dry pyridine (2 ml),and dehydrated by evaporation under reduced pressure. This procedure wasrepeated three times. To this, 4% tetrazole solution in acetonitrile (15ml) was added, and the mixture was stirred at room temperature. Thereaction was monitored by silica gel thin layer. chromatography (TLC,developing solvent: chloroform:methanol=9:1). The reaction was usuallyfinished in a day. After the reaction, the solvent was removed underreduced pressure. To the residue, 0.1 M solution of iodine intetrahydrofuran/pyridine/water (80:40:2, 3 ml) was added, and the formedphosphite triester was oxidized with stirring at room temperature. Oneand a half hours later, the solvent was removed under reduced pressure,and the residue was extracted with chloroform. The extract was driedover anhydrous magnesium sulfate, and the solvent was removed underreduced pressure. The residue was subjected to silica gel columnchromatography, and eluted with chloroform/methanol=90:10. Theribocytidylpuromycin (CpPur) having protection groups was eluted bysilica gel TLC (developing solvent; chloroform:methanol=9:1) at an Rf of0.32. Then, protection groups were removed. The ribocytidylpuromycinhaving protecting groups was first treated with 80% aqueous solution ofacetic acid (0.5 ml) at room temperature for 1 hour. After the aceticacid was removed under reduced pressure, to the residue, a mixedsolution of aqueous ammonia/ethanol=2:1 (0.5 ml) was added. After themixture was left at room temperature for 15 hours, the solvent wasremoved under reduced pressure, and to the residue, 1 M solution oftetrabutylammonium fluoride in tetrahydrofuran (0.5 ml) was added toremove β-cyanoethyl group. Thirty minutes later, the solvent was removedunder reduced pressure, and the residue was subjected to columnchromatography using QAE-Sephadex, and eluted with 0 to 0.5 M lineargradient of triethylamine carbonate. The eluent was collected andlyophilized to afford 10 mg of ribocytidylpuromycin. The synthesizedproduct was confirmed to be ribocytidylpuromycin by the facts that itafforded equimolar amounts of cytidine and puromycin-5′-phosphate afterdigestion with nuclease P1, and that molecular ions of [M+H]⁺ wereappeared at m/z 777 in MALDI/TOF mass spectrometry.

[0158] <2> Nucleic Acid Portion (Preparation of in vitro Virus Genome)

[0159] Materials:

[0160] A cell-free protein synthesis system of rabbit reticulocytelysate (Nuclease treated Rabbit reticulocyte lysate) was purchased fromPromega. T7 RNA polymerase, T4 DNA ligase, T4 RNA ligase, T4polynucleotide kinase, human placenta ribonuclease inhibitor, EcoRI,BamHI, and deoxyribonucleotides were purchased from Takara Shuzo.Restriction enzymes BstNI, and BglII were purchased from New EnglandLabs. As for [³⁵S]-methionine, and [³²P]-γATP, those from Amersham, andas for Taq DNA polymerase, those from Kurabo and Grainer were used. Asfor the other biochemical reagents, those from Sigma and Wako PureChemicals were used. A plasmid containing the N-terminal half region ofhuman tau protein (amino acid residue numbers 1-165) (pAR3040) wasprepared by picking up the full length gene of human tau protein by PCRmethod from a cDNA library of human brain cloned in λZAPII, introducingthe gene into a plasmid, amplifying only the N-terminal half region byPCR, and introducing the amplified product into a plasmid. As the PCR(polymerase chain reaction) apparatus, Model ASTEC PC800 (Astec) wasused.

[0161] (1) Preparation of Genome

[0162] A. Preparation of DNA for N-terminal Half Region

[0163] mRNAs encoding the N-terminal half region of human tau protein(amino acid residues 1-165) with or without stop codon, which wereligated with spacer, peptide acceptor, and rCpPur at its 3′-terminalend, were constructed as follows (FIG. 8).

[0164] 1) A plasmid into which the N-terminal half region of human tauprotein (Goedert, M. (1989) EMBO J. 8, 392-399) (pAR3040) wasintroduced, was linearized by digestion with a restriction enzyme BglII.

[0165] 2) The N-terminal half region (amino acid residue numbers 1-165)was amplified from the above genome by PCR. As primers, used were Leftl(SEQ ID NO: 18) for the 5′ side, and Right1 (SEQ ID NO: 19) with stopcodon, or Right2 (SEQ ID NO: 20) without stop codon for the 3′ side. ThePCR conditions consisted of 92° C./30 seconds for denaturation, 65°C./30 seconds for annealing, and 73° C./1 minute for elongation, andthis cycle was repeated 30 times.

[0166] 3) A DNA sequence composed of the promoter region of T7 RNApolymerase, Kozak sequence, and DNA sequence corresponding to amino acidresidue numbers 1-25 of human tau protein connected in this order (SEQID NO: 21) was prepared by chemical synthesis.

[0167] 4) The two kinds of purified DNA obtained in the procedures ofthe above 2) and 3) were connected by a two-step PCR as follows. Thatis, a mixture of the aforementioned two kinds of DNA was first amplifiedin the absence of primer, and subsequently amplified in the presence ofprimers of Left2 (SEQ ID NO: 22) and Right1 (SEQ ID NO: 19) or Right2(SEQ ID NO: 20). From the above procedure, a DNA containing the promoterof T7 RNA polymerase and Kozak sequence at an upstream side of ORF ofthe N-terminal half region of human tau protein was prepared. An RNA wasobtained through a reaction using this DNA as template, and T7 RNApolymerase at 37° C. for two hours.

[0168] B. Ligation of Spacer and Peptide Acceptor

[0169] Spacer 5 (SEQ ID NO: 23) and a peptide acceptor (P-Acceptor, SEQID NO: 15), which was a chimeric nucleic acid composed of 21-nucleotideDNA and 4-nucleotide RNA, i.e., 25 nucleotides in total, was chemicallysynthesized. The 5′-terminal end of the peptide acceptor wasphosphorylated by reaction at 36° C. for 1 hour using T4 polynucleotidekinase, and the peptide acceptor was backed with a splint DNA (SEQ IDNO: 24) having a sequence complementary thereto, and ligated to the3′-terminal end of Spacer 5 through a reaction at 16° C. for 2 hoursusing T4 DNA ligase.

[0170] C. Ligation of RNA and Spacer-peptide Acceptor

[0171] The ligation product of Spacer 5-peptide acceptor obtained in theabove B was phosphorylated at the 5′-terminal end through a reaction at36° C. for 1 hour using T4 polynucleotide kinase, and ligated to the RNAobtained in the above A through a reaction at 4° C. for 48 hours usingT4 RNA ligase.

[0172] D. Ligation of rCpPur

[0173] The rCpPur obtained in the above <1> Preparation of 3′-terminalend portion of nucleic acid portion was phosphorylated through areaction at 15° C. for 24 hours using T4 polynucleotide kinase, andligated to the 3′-terminal end of the genome prepared in the above Cthrough a reaction at 37° C. for 30 minutes using T4 RNA ligase. Fromthis procedure, a chimera RNA genome having puromycin at its 3′-terminalend could be constructed.

[0174] E. Bonding of rCpPur to C-terminal End of N-terminal Half ofHuman tau Protein

[0175] It is considered that, in obtaining effective binding of aC-terminal end of a protein and an RNA encoding it, the distance betweenpuromycin and a stop codon, and presence or absence of a stop codonwould become important factors. Therefore, in order to examine effectsof these factors, the following three kinds of genomes, mRNAs encodingthe N-terminal half of human tau protein each (1) having a stop codonbut not having a DNA spacer, (2) having neither of a stop codon and aDNA spacer, and (3) not having a stop codon but having a DNA spacer, atthe 3′-terminal end were prepared. By using these three kinds ofgenomes, protein synthesis was performed in the presence of rCpPurlabeled with ³²P in a cell-free translation system utilizing rabbitreticulocyte lysate (FIG. 9). It was found that, when the 3′-terminalend did not have a DNA spacer, rCpPur was bound to the C-terminal of theprotein with a similar efficiency regardless of the presence or absenceof a stop codon. That is, in SDS-PAGE (SDS-polyacrylamide gelelectrophoresis), the bands of the proteins bonded to rCpPur (the firstand the second lanes from the left in FIG. 9) appeared at the samelocation as that of the protein monomer labeled with [³⁵S]-methionine(the most right lane in FIG. 9). On the other hand, if mRNA had a DNAspacer, rCpPur was bonded to the C-terminal end of the protein at anefficiency about three times higher than those obtained in the formertwo kinds of mRNA even without a stop codon (the third lane from theleft, FIG. 9). This result can be considered to indicate thattranslation pausing of ribosome occurred on the DNA sequence, and as aresult, rCpPur and the protein could be bonded efficiently. Further,this result suggests that, when a genome without a stop codon having aDNA spacer and rCpPur at its 3′-terminal end is used as mRNA in acell-free translation system, puromycin at the 3′-terminal end of mRNAcan efficiently be bonded to the C-terminal end of the correspondingtranslated protein.

[0176] <3> Construction of in vitro Virus in Cell-free TranslationSystem

[0177] The genome constructed in the above <2> Nucleic acid portion(preparation of in vitro virus genome) composed of mRNA encoding theN-terminal half of human tau protein (1-165), DNA spacer (105 mer),peptide acceptor and rCpPur was translated by using rabbit reticulocytelysate. When incorporation of [³⁵S]-methionine into the protein wasexamined first by using an RNA encoding the N-terminal half (1-165) ofhuman tau protein as mRNA, bands appeared at locations corresponding tomonomer (about 28 KDa) and dimer (about 55 KDa) of the N-terminal half(1-165). In this case, the monomer was the major product, and the dimerwas observed in an extremely small amount (the first left lane in FIG.10(A)). This result indicates that the RNA encoding the N-terminal halfof human tau protein (1-165) functioned as mRNA. When the genomecomposed of mRNA encoding the N-terminal half (1-165) of human tauprotein, DNA spacer (105 mer), peptide acceptor and rCpPur wastranslated in a similar cell-free translation system containing[³⁵S]-methionine, and the products were analyzed in time course (5minutes, 10 minutes, 20 minutes, and 40 minutes), a new wide bandappeared at a location slightly above that of the genome in addition tothe bands at the location of the monomer and dimer (the first right lanein FIG. 10(A)). The intensity of this band increased with increase ofreaction time (the second to fifth lanes from the left in FIG. 10(A)),and increase of the genome amount (Lanes 3 and 4 in FIG. 10(B)). Theseresults indicate that the genome was bonded to the C-terminal end of theprotein with a covalent bond through puromycin. This also means that agenotype was covalently bound to a phenotype. That is, a molecule thatassigns a genotype to a phenotypes was formed. The present inventorsdesignated this assigning molecule as in vitro virus. When the effect ofthe length of DNA spacer on the formation of in vitro virus wasexamined, it was found that the in vitro virus was not formedefficiently with a length of about 80 mer, and it required a length ofat least 100 mer.

[0178] Further, the generation of in vitro virus was confirmed by usingrcpPur labeled with ³²P. That is, a genome composed of mRNA encoding theN-terminal half (1-165) of human tau protein, DNA spacer (105 mer),peptide acceptor, and [³²P]-rCpPur was translated by using rabbitreticulocyte lysate. The bonding of the genome and the protein wasconfirmed by digestion with mung bean nuclease. That is, when thetranslation product (Lane 3 in FIG. 11) was digested with mung beannuclease, bands appeared at the locations corresponding to monomer anddimer (Lane 1 in FIG. 11) of the N-terminal half (1-165) of human tauprotein (Lane 4 in FIG. 11). This indicates that the rCpPur labeled with³²P was attached to the 3′-terminal end of the protein. Also from thisresult, it was confirmed that the genome was bound to the C-terminal endof the protein with a covalent bond through puromycin. The efficiency ofthe binding was estimated to be about 10%. Because in vitro virus genomehaving a concentration of 40 to 100 pmol/ml can be prepared, generatedin vitro viruses would consist of a population containing 2.4 to 6×10¹²of mutants, and this number corresponds to 10000 times of that obtainedin the phage display method (Scott, J. K. & Smith, G. P. (1990) Science249, 386-390). The genotype assignment to phenotype has advantages, forexample, it eliminates the problem concerning the permeability, and itenables incorporation of various non-naturally-occurring amino acids,and therefore it enables synthesis of an extremely large number ofmutants, or creation of various functional proteins.

Example 3

[0179] Protein Evolution Simulation Method Utilizing in vitro Viruses

[0180] The protein evolution simulation method utilizing in vitroviruses comprises, as shown in FIG. 12, (1) construction of in vitrovirus genomes, (2) completion of in vitro viruses, (3) selectionprocess, (4) introduction of mutation, and (5) amplification, and itallows modification and creation of functional proteins. In particular,repetition of these steps allows efficient modification and creation offunctional proteins. Among these steps, the steps of (1) and (2) werespecifically explained in Examples 1 and 2 mentioned above. Therefore,the steps (3), (4) and (5) will be explained inthis example.

[0181] First, it was examined whether peptides specific to an antibodywere selected. Specifically, mouse IgG was used as the antibody, and theknown ZZ region of protein A (Nilsson, B., et al., (1987) Protein Eng.,1, 107-113) was used as a peptide sequence to be specifically bound tothe antibody. As a control, N-terminal region (1-105) of human tauprotein (Goedert, M. (1989) EMBO J. 8, 392-399) was used. According tothe construction methods of in vitro viruses described in the aboveExamples 1 and 2, in vitro virus genomes encoding the ZZ region ofprotein A and the N-terminal region (1-105) of human tau protein wereprepared. With different ratios of the in vitro virus genome encodingthe ZZ region of protein A and the in vitro virus genome encoding theN-terminal region (1-105) of human tau protein varying as 1:1, 1:10,1:100, 1:1000 or the like, they were translated in a cell-freetranslation system utilizing rabbit reticulocyte lysate at 30° C. for 10minutes. Then, the translation product was diluted, and centrifuged toremove insoluble fractions, and the supernatant was added to amicroplate coated with mouse IgG (blocked with bovine serum albuminbeforehand), and left stand at 4° C. for 2 hours. Then, the translationproduct was removed from the microplate, and it was washed with awashing buffer (50 mM Tris/acetic acid, pH 7.5, 150 mM NaCl, 10 mM EDTA,0.1% Tween 20) for 6 times in total, and eluted 2 times with an elutionbuffer (1 M acetic acid, pH 2.8). The eluted solution was subjected toethanol precipitation, and the precipitates were dissolved in sterilewater (20 μl), and used as a template for reverse transcription PCR. Thereverse transcription PCR was performed by using reverse transcriptase(Avian Mieloblastosis virus Reverse Transcriptase, Promega), DNApolymerase (Tfl DNA Polymerase, Promega), and RT+ (SEQ ID NO: 25) andRT− (SEQ ID NO: 26) as primers. Following a reaction at 48° C. for 40minutes, the reverse transcriptase was inactivated with a treatment at94° C. for 5 minutes, and a cycle of 94° C. for 30 seconds, 66° C. for40 seconds, and 72° C. for 40 seconds was repeated 30 times. Theobtained PCR product was electrophoresed at 55° C. on 4% polyacrylamidegel containing 8 M urea, and observed by silver staining. As a result,it was found that the in vitro virus genome containing the ZZ region ofprotein A could be amplified even in an amount of one-100th of thecontrol genome, i.e., the in vitro virus genome containing theN-terminal region (1-105) of human tau protein. This result indicatesthat the in vitro virus genome containing the ZZ region of protein A wasspecifically bound to mouse IgG through the translated ZZ region ofprotein A. Therefore, it was confirmed that the in vitro viruses couldbe selected. Introduction of mutation and amplification can be performedby using the already-established error-prone PCR (Leung, D. W., et al.,(1989) J. Methods Cell Mol. Biol., 1, 11-15), Sexual PCR (Stemmer, W. P.C. (1994) Proc. Natl. Acad. Sci. If USA 91, 10747-10751) or the like.Therefore, it was verified that the protein evolution simulation methodshown in FIG. 12 was feasible.

[0182] Industrial Applicability

[0183] According to the present invention, a molecule assigning agenotype (nucleic acid portion) to a phenotype (protein portion), andconstruction methods therefor are provided. There are also provided aprotein evolution simulation method utilizing molecules that assign agenotype to a phenotype (in vitro viruses) constructed according to thepresent invention, which comprises selecting the in vitro viruses by thein vitro selection method, amplifying the gene portion of an extremelysmall amount of the selected in vitro viruses by reverse transcriptionPCR, and further performing amplification while introducing a mutation,and the like. The molecule assigning the genotype to the phenotype, theprotein evolution simulation method utilizing it and the like of thepresent invention are an extremely useful substance or experimentalsystem for evolutionary molecular engineering, i.e., modification offunctional biopolymers such as enzymes, antibodies, and ribozymes, andcreation of biopolymers having functions which cannot be found in livingorganisms.

1 26 33 nucleic acid single linear other nucleic acid synthetic DNA T7promoter upstream 1 GAGCATAGAT CTCGATCCCG CGAAATTAAT ACG 33 33 nucleicacid single linear other nucleic acid synthetic DNA includes atermination codon 2 GCAGCCGGAT CCTTACTACT TGTGGGTTTC AAT 33 33 nucleicacid single linear other nucleic acid synthetic DNA includes aninitiation codon; complementary to SEQ ID NO 4 3 GGACATGACA TTCATCATGTCTGGCATATG TAT 33 33 nucleic acid single linear other nucleic acidsynthetic DNA includes an initiation codon; complementary to SEQ ID NO 34 ATACATATGC CAGACATGAT GAATGTCATG TCC 33 16 nucleic acid single linearother nucleic acid synthetic DNA has a portion complementary to SEQ IDNO 6 5 GATCTATTTC TTATTC 16 17 nucleic acid single linear other nucleicacid synthetic DNA has an initiation codon; complementary to SEQ ID NO 56 GAAGAGAATA AGAAATA 17 17 nucleic acid single linear other nucleic acidsynthetic DNA has a portion complementary to SEQ ID NO 6 7 TCTTCTATTTCTTATTC 17 30 nucleic acid single linear other nucleic acid syntheticDNA has a portion complementary to SEQ ID NO 9 8 GGGTAAACGA ATGAACAAGAATAAGAAATA 30 108 nucleic acid single linear other nucleic acidsynthetic DNA has a sequence of an alanyl tRNA 9 TTGTTCATTC GTTTACCCGGGGCTATAGCT CAGCTGGGAG AGCGCCTGCT TCTAAC 60 GAGGTCTGCG GTTCGATCCCGCGTAGCTCC ACCAGGAGGC GACTAGCT 108 23 nucleic acid single linear othernucleic acid synthetic DNA has a 3′-side sequence of an alanyl tRNA 10GTGGAGCTAC GCGGGATCGA ACC 23 25 nucleic acid single linear other nucleicacid synthetic DNA has no initiation codon 11 GCAGCCGGAT CCTTTCTGCTTGTGG 25 21 nucleic acid single linear other nucleic acid synthetic DNAhas a sequence partly complementary to SEQ ID NO 17 12 CTTTAATGACCTCCCCTCTC C 21 40 nucleic acid single linear other nucleic acidsynthetic DNA has a sequence partly complementary to SEQ ID NO 17 13CTTTAATAAT TTTTTTTTTT TTTAATGACC TCCCCTCTCC 40 60 nucleic acid singlelinear other nucleic acid synthetic DNA has a sequence partlycomplementary to SEQ ID NO 17 14 CTTTAATAAT TTTTTTTTTT TTTTTTTTTTTTTTTTTTTT TTTAATGACC TCCCCT 60 80 nucleic acid single linear othernucleic acid synthetic DNA has a sequence partly complementary to SEQ IDNO 17 15 CTTTAATAAT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTT60 TTTAATGACC TCCCCTCTCC 80 25 nucleic acid single linear other nucleicacid synthetic DNA 22..25 RNA 16 CTTACTGTCT TTTTTTTTTT TGAGC 25 33nucleic acid single linear other nucleic acid synthetic DNA has asequence partly complementary to SEQ ID NO 16 17 AAAAAAGACA GTAAGGGAGAGGGGAGGTCA TTA 33 24 nucleic acid single linear other nucleic acidsynthetic DNA includes an N-terminal initiation codon in anN-terminal-half region of a tau protein 18 ATGGCTGAGC CCCGCATGGA GTTC 2424 nucleic acid single linear other nucleic acid synthetic DNA includesa C-terminal termination codon in an N-terminal-half region of a tauprotein 19 CTCTGCCACT TACTAGGGCT CCCG 24 23 nucleic acid single linearother nucleic acid synthetic DNA includes a C-terminal termination codonin an N-terminal-half region of a tau protein 20 CTCTGCCACC TTCTTGGGCTCCC 23 118 nucleic acid single linear other nucleic acid synthetic DNAincludes a promoter region of T7 RNA polymerase, a kozak sequence, and aDNA sequence corresponding to amino acid numbers 1-25 of a human tauprotein 21 GATCCCGCGA AATTAATACG ACTCACTATA GGGAGACCAC AACGGTTTCC CTCTAG60 AATTTTGTTT AACTTTAAGA AGGAGATGCC ACCATGGTTG AGCCCCGCAT GGAGT 118 30nucleic acid single linear other nucleic acid synthetic DNA a 5′-endregion of T7 RNA polymerase including a part of a promoter thereof 22GATCCCGCGA AATTAATACG ACTCACTATA 30 105 nucleic acid single linear othernucleic acid synthetic DNA Spacer 5 23 AAGCCACTCG CGTGGTCTCG CATTTTTTTTTTTTTTTTTT TTTTTTTTTT TTTTTT 60 TTTTTTTTTT TTTTTTTTTT TTTTTTTTAATGACCTCCCC TCTCC 105 25 nucleic acid single linear other nucleic acidsynthetic DNA splint DNA 24 AAAGACAGTA AGGGAGAGGG GAGGT 25 47 nucleicacid single linear other nucleic acid synthetic DNA a primer for reversetranscription PCR 25 GGTTTCCCTC TAGAAATAAT TTTGTTTAAC TTTAAGAAGG AGATATA47 25 nucleic acid single linear other nucleic acid synthetic DNA aprimer for reverse transcription PCR 26 AGCTTTCAGG CCAGCGTCCG TGTCA 25

What is claimed is:
 1. A molecule assigning a genotype to a phenotype,which comprises a nucleic acid portion having a nucleotide sequencereflecting the genotype, and a protein portion comprising a proteininvolved in exhibition of the phenotype, the nucleic acid portion andthe protein portion being directly bound by a chemical bond.
 2. Theassigning molecule according to claim 1, wherein a 3′-terminal end ofthe nucleic acid portion and a C-terminal end of the protein portion arebonded with a covalent bond.
 3. The assigning molecule according toclaim 1 or 2, wherein a 3′-terminal end of the nucleic acid portioncovalently bonded to a C-terminal end of the protein portion ispuromycin.
 4. The assigning molecule according to any one of claims 1 to3, wherein the nucleic acid portion comprises a gene encoding a protein,and the protein portion is a translation product of the gene of thenucleic acid portion.
 5. The assigning molecule according to claim 4,wherein the nucleic acid portion comprises a gene composed of RNA, and asuppressor tRNA bonded to the gene through a spacer.
 6. The assigningmolecule according to claim 5, wherein the suppressor tRNA comprises ananticodon corresponding to a termination codon of the gene.
 7. Theassigning molecule according to claim 4, wherein the nucleic acidportion comprises a gene composed of RNA, and a spacer composed of DNAand RNA.
 8. The assigning molecule according to claim 4, wherein thenucleic acid portion comprises a gene composed of RNA, and a spacercomposed of DNA and polyethylene glycol.
 9. The assigning moleculeaccording to claim 4, wherein the nucleic acid portion comprises a genecomposed of RNA, and a spacer composed of a double-stranded DNA.
 10. Theassigning molecule according to claim 4, wherein the nucleic acidportion comprises a gene composed of RNA, and a spacer composed of adouble strand of RNA and a short chain peptide nucleic acid (PNA) orDNA.
 11. The assigning molecule according to claim 4, wherein thenucleic acid portion comprises a gene composed of DNA, and a spacercomposed of DNA and RNA.
 12. A method for constructing the assigningmolecule as defined in claim 5, which comprises (a) bonding a DNAcomprising a sequence corresponding to a suppressor tRNA, to a3′-terminal end of a DNA containing a gene through a spacer, (b)transcribing the obtained DNA bonded product into RNA, (c) bonding, to a3′-terminal end of the obtained RNA, a nucleoside or a substance havinga chemical structure analogous to that of a nucleoside, which can becovalently bound to an amino acid or a substance having a chemicalstructure analogous to that of an amino acid, and (d) performing proteinsynthesis in a cell-free protein synthesis system using the obtainedbonded product as mRNA to bond a nucleic acid portion containing thegene to a translation product of the gene.
 13. A method for constructingthe assigning molecule as defined in claim 7, which comprises (a)preparing a DNA containing a gene which has no termination codon, (b)transcribing the prepared DNA into RNA, (c) bonding a chimeric spacercomposed of DNA and RNA to a 3′-terminal end of the obtained RNA, (d)bonding, to a 3′-terminal end of the obtained bonded product, anucleoside or a substance having a chemical structure analogous to thatof a nucleoside, which can be covalently bound to an amino acid or asubstance having a chemical structure analogous to that of an aminoacid, and (e) performing protein synthesis in a cell-free proteinsynthesis system using the obtained bonded product as mRNA to bond anucleic acid portion containing the gene to a translation product of thegene.
 14. The construction method according to claim 12 or 13, whereinthe nucleoside or the substance having the chemical structure analogousto that of the nucleoside is puromycin.
 15. A method for constructingthe assigning molecule as defined in claim 8, which comprises (a)preparing a DNA containing a gene which has no termination codon, (b)transcribing the prepared DNA into RNA, (c) bonding a chimeric spacercomposed of DNA and polyethylene glycol to a 3′-terminal end of theobtained RNA, (d) bonding, to a 3′-terminal end of the obtained bondedproduct, a nucleoside or a substance having a chemical structureanalogous to that of a nucleoside, which can be covalently bound to anamino acid or a substance having a chemical structure analogous to thatof an amino acid, and (e) performing protein synthesis in a cell-freeprotein synthesis system using the obtained bonded product as mRNA tobond a nucleic acid portion containing the gene to a translation productof the gene.
 16. A method for constructing the assigning molecule asdefined in claim 9, which comprises (a) preparing a DNA containing agene which has no termination codon, (b) transcribing the prepared DNAinto RNA, (c) bonding a spacer composed of double-stranded DNA to a3′-terminal end of the obtained RNA, (d) bonding, to a 3′-terminal endof the obtained bonded product, a nucleoside or a substance having achemical structure analogous to that of a nucleoside, which can becovalently bound to an amino acid or a substance having a chemicalstructure analogous to that of an amino acid, and (e) performing proteinsynthesis in a cell-free protein synthesis system using the obtainedbonded product as mRNA to bond a nucleic acid portion containing thegene to a translation product of the gene.
 17. A method for constructingthe assigning molecule as defined in claim 10, which comprises (a)preparing a DNA containing a gene which has no termination codon, and anucleotide sequence of a spacer, (b) transcribing the prepared DNA intoRNA, (c) bonding, to a 3′-terminal end of the obtained RNA, a nucleosideor a substance having a chemical structure analogous to that of anucleoside, which can be covalently bound to an amino acid or asubstance having a chemical structure analogous to that of an aminoacid, (d) adding a short chain PNA or DNA to a 3′-terminal end sideportion of the gene in the obtained RNA bonded product to form adouble-stranded chain, and (e) performing protein synthesis in acell-free protein synthesis system using the obtained bonded product asmRNA to bond a nucleic acid portion containing the gene to a translationproduct of the gene.
 18. A method for protein evolution simulation,which comprises a construction step for constructing assigning moleculesfrom a DNA containing a gene by the construction method as defined inany one of claims 12, 13, 15, 16 and 17, a selection step for selectingthe assigning molecules obtained in the construction step, a mutationintroduction step for introducing a mutation into a gene portion of anassigning molecule selected in the selection step, and an amplificationstep for amplifying the gene portion obtained in the mutationintroduction step.
 19. The method for protein evolution simulationaccording to claim 18, wherein the construction step, the selectionstep, the mutation introduction step and the amplification step arerepeatedly performed by providing the DNA obtained in the amplificationstep to the construction step.
 20. A method for assaying protein/proteinor protein/nucleic acid intermolecular action, which comprises aconstruction step for constructing assigning molecules by theconstruction method of any one as defined in claims 12, 13, 15, 16 and17, and an assay step for examining intermolecular action of theassigning molecules obtained in the construction step with anotherprotein or nucleic acid.
 21. An apparatus for performing the method forevolution simulation as defined in claim 18 or 19, which comprises ameans for constructing assigning molecules, said means comprising afirst bonding means for bonding a DNA comprising a sequencecorresponding to a suppressor tRNA to a 3′-terminal end of a DNAcontaining a gene through a spacer, a transcription means fortranscribing the DNA bonded product obtained by the first bonding meansinto RNA, a second bonding means for bonding, to a 3′-terminal end ofthe RNA obtained by the transcription means, a nucleoside or a substancehaving a chemical structure analogous to that of a nucleoside, which canbe covalently bound to an amino acid or a substance having a chemicalstructure analogous to that of an amino acid, and a third bonding meansfor performing protein synthesis in a cell-free protein synthesis systemusing the bonded product obtained by the second bonding means as mRNA tobond a nucleic acid portion containing the gene to a translation productof the gene, or a means for constructing assigning molecules, said meanscomprising a transcription means for transcribing a DNA containing agene into RNA, a first bonding means for bonding a chimeric spacercomposed of DNA and RNA, a chimeric spacer composed of DNA andpolyethylene glycol, a double-stranded spacer composed of DNA, or adouble-stranded spacer composed of RNA and PNA or DNA to a 3′-terminalend of the RNA obtained by the transcription means, a second bondingmeans for bonding, to a 3′-terminal end of the RNA-spacer bonded productobtained by the first bonding means, a nucleoside or a substance havinga chemical structure analogous to that of a nucleoside, which can becovalently bound to an amino acid or a substance having a chemicalstructure analogous to that of an amino acid, and a third bonding meansfor performing protein synthesis in a cell-free protein synthesis systemusing the bonded product obtained by the second bonding means as mRNA tobond a nucleic acid portion containing the gene to a translation productof the gene; a selection means for selecting the constructed assigningmolecules; a mutation introduction means for introducing a mutation intoa gene portion of an assigning molecule selected; and an amplificationmeans for amplifying the gene portion to which the mutation isintroduced.